Human OTULIN haploinsufficiency impairs cell-intrinsic immunity to staphylococcal α-toxin.

The molecular basis of interindividual clinical variability upon infection with Staphylococcus aureus is unclear. We describe patients with haploinsufficiency for the linear deubiquitinase OTULIN, encoded by a gene on chromosome 5p. Patients suffer from episodes of life-threatening necrosis, typically triggered by S. aureus infection. The disorder is phenocopied in patients with the 5p- (Cri-du-Chat) chromosomal deletion syndrome. OTULIN haploinsufficiency causes an accumulation of linear ubiquitin in dermal fibroblasts, but tumor necrosis factor receptor-mediated nuclear factor κB signaling remains intact. Blood leukocyte subsets are unaffected. The OTULIN-dependent accumulation of caveolin-1 in dermal fibroblasts, but not leukocytes, facilitates the cytotoxic damage inflicted by the staphylococcal virulence factor α-toxin. Naturally elicited antibodies against α-toxin contribute to incomplete clinical penetrance. Human OTULIN haploinsufficiency underlies life-threatening staphylococcal disease by disrupting cell-intrinsic immunity to α-toxin in nonleukocytic cells.

Primaquine as a Candidate for HHV-8-Associated Primary Effusion Lymphoma and Kaposi's Sarcoma Treatment.

Human Herpesvirus 8 (HHV-8) is associated with three main severe orphan malignancies, Kaposi's sarcoma (KS), multicentric Castleman's disease (MCD), and primary effusion lymphoma (PEL), which present few therapeutic options. We identified the antimalarial primaquine diphosphate (PQ) as a promising therapeutic candidate for HHV-8-associated PEL and KS. Indeed, PQ strongly reduced cell viability through caspase-dependent apoptosis, specifically in HHV-8-infected PEL cells. Reactive oxygen species (ROS)- and endoplasmic reticulum (ER) stress-mediated apoptosis signaling pathways were found to be part of the in vitro cytotoxic effect of PQ. Moreover, PQ treatment had a clinically positive effect in a nonobese diabetic (NOD)/SCID xenograft PEL mouse model, showing a reduction in tumor growth and an improvement in survival. Finally, an exploratory proof-of-concept clinical trial in four patients harboring severe KS was conducted, with the main objectives to assess the efficacy, the safety, and the tolerability of PQ, and which demonstrated a positive efficacy on Kaposi's sarcoma-related lesions and lymphedema.

Role of Optineurin in the Mitochondrial Dysfunction: Potential Implications in Neurodegenerative Diseases and Cancer.

Optineurin (Optn) is a 577 aa protein encoded by the Optn gene. Mutations of Optn are associated with normal tension glaucoma and amyotrophic lateral sclerosis, and its gene has also been linked to the development of Paget's disease of bone and Crohn's disease. Optn is involved in diverse cellular functions, including NF-κB regulation, membrane trafficking, exocytosis, vesicle transport, reorganization of actin and microtubules, cell cycle control, and autophagy. Besides its role in xenophagy and autophagy of aggregates, Optn has been identified as a primary autophagy receptor, among the five adaptors that translocate to mitochondria during mitophagy. Mitophagy is a selective macroautophagy process during which irreparable mitochondria are degraded, preventing accumulation of defective mitochondria and limiting the release of reactive oxygen species and proapoptotic factors. Mitochondrial quality control via mitophagy is central to the health of cells. One of the important surveillance pathways of mitochondrial health is the recently defined signal transduction pathway involving the mitochondrial PTEN-induced putative kinase 1 (PINK1) protein and the cytosolic RING-between-RING ubiquitin ligase Parkin. Both of these proteins, when mutated, have been identified in certain forms of Parkinson's disease. By targeting ubiquitinated mitochondria to autophagosomes through its association with autophagy related proteins, Optn is responsible for a critical step in mitophagy. This review reports recent discoveries on the role of Optn in mitophagy and provides insight into its link with neurodegenerative diseases. We will also discuss the involvement of Optn in other pathologies in which mitophagy dysfunctions are involved including cancer.

Super-resolution microscopy reveals a preformed NEMO lattice structure that is collapsed in incontinentia pigmenti.

The NF-κB pathway has critical roles in cancer, immunity and inflammatory responses. Understanding the mechanism(s) by which mutations in genes involved in the pathway cause disease has provided valuable insight into its regulation, yet many aspects remain unexplained. Several lines of evidence have led to the hypothesis that the regulatory/sensor protein NEMO acts as a biological binary switch. This hypothesis depends on the formation of a higher-order structure, which has yet to be identified using traditional molecular techniques. Here we use super-resolution microscopy to reveal the existence of higher-order NEMO lattice structures dependent on the presence of polyubiquitin chains before NF-κB activation. Such structures may permit proximity-based trans-autophosphorylation, leading to cooperative activation of the signalling cascade. We further show that NF-κB activation results in modification of these structures. Finally, we demonstrate that these structures are abrogated in cells derived from incontinentia pigmenti patients.

Genetic deletion of keratin 8 corrects the altered bone formation and osteopenia in a mouse model of cystic fibrosis.

Patients with cystic fibrosis (CF) display low bone mass and alterations in bone formation. Mice carrying the F508del genetic mutation in the cystic fibrosis conductance regulator (Cftr) gene display reduced bone formation and decreased bone mass. However, the underlying molecular mechanisms leading to these skeletal defects are unknown, which precludes the development of an efficient anti-osteoporotic therapeutic strategy. Here we report a key role for the intermediate filament protein keratin 8 (Krt8), in the osteoblast dysfunctions in F508del-Cftr mice. We found that murine and human osteoblasts express Cftr and Krt8 at low levels. Genetic studies showed that Krt8 deletion (Krt8(-/-)) in F508del-Cftr mice increased the levels of circulating markers of bone formation, corrected the expression of osteoblast phenotypic genes, promoted trabecular bone formation and improved bone mass and microarchitecture. Mechanistically, Krt8 deletion in F508del-Cftr mice corrected overactive NF-κB signaling and decreased Wnt-ß-catenin signaling induced by the F508del-Cftr mutation in osteoblasts. In vitro, treatment with compound 407, which specifically disrupts the Krt8-F508del-Cftr interaction in epithelial cells, corrected the abnormal NF-κB and Wnt-ß-catenin signaling and the altered phenotypic gene expression in F508del-Cftr osteoblasts. In vivo, short-term treatment with 407 corrected the altered Wnt-ß-catenin signaling and bone formation in F508del-Cftr mice. Collectively, the results show that genetic or pharmacologic targeting of Krt8 leads to correction of osteoblast dysfunctions, altered bone formation and osteopenia in F508del-Cftr mice, providing a therapeutic strategy targeting the Krt8-F508del-CFTR interaction to correct the abnormal bone formation and bone loss in cystic fibrosis.

Increased NF-κB Activity and Decreased Wnt/ß-Catenin Signaling Mediate Reduced Osteoblast Differentiation and Function in ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mice.

The prevalent human ΔF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is associated with reduced bone formation and bone loss in mice. The molecular mechanisms by which the ΔF508-CFTR mutation causes alterations in bone formation are poorly known. In this study, we analyzed the osteoblast phenotype in ΔF508-CFTR mice and characterized the signaling mechanisms underlying this phenotype. Ex vivo studies showed that the ΔF508-CFTR mutation negatively impacted the differentiation of bone marrow stromal cells into osteoblasts and the activity of osteoblasts, demonstrating that the ΔF508-CFTR mutation alters both osteoblast differentiation and function. Treatment with a CFTR corrector rescued the abnormal collagen gene expression in ΔF508-CFTR osteoblasts. Mechanistic analysis revealed that NF-κB signaling and transcriptional activity were increased in mutant osteoblasts. Functional studies showed that the activation of NF-κB transcriptional activity in mutant osteoblasts resulted in increased ß-catenin phosphorylation, reduced osteoblast ß-catenin expression, and altered expression of Wnt/ß-catenin target genes. Pharmacological inhibition of NF-κB activity or activation of canonical Wnt signaling rescued Wnt target gene expression and corrected osteoblast differentiation and function in bone marrow stromal cells and osteoblasts from ΔF508-CFTR mice. Overall, the results show that the ΔF508-CFTR mutation impairs osteoblast differentiation and function as a result of overactive NF-κB and reduced Wnt/ß-catenin signaling. Moreover, the data indicate that pharmacological inhibition of NF-κB or activation of Wnt/ß-catenin signaling can rescue the abnormal osteoblast differentiation and function induced by the prevalent ΔF508-CFTR mutation, suggesting novel therapeutic strategies to correct the osteoblast dysfunctions in cystic fibrosis.

Human HOIP and LUBAC deficiency underlies autoinflammation, immunodeficiency, amylopectinosis, and lymphangiectasia.

Inherited, complete deficiency of human HOIL-1, a component of the linear ubiquitination chain assembly complex (LUBAC), underlies autoinflammation, infections, and amylopectinosis. We report the clinical description and molecular analysis of a novel inherited disorder of the human LUBAC complex. A patient with multiorgan autoinflammation, combined immunodeficiency, subclinical amylopectinosis, and systemic lymphangiectasia, is homozygous for a mutation in HOIP, the gene encoding the catalytic component of LUBAC. The missense allele (L72P, in the PUB domain) is at least severely hypomorphic, as it impairs HOIP expression and destabilizes the whole LUBAC complex. Linear ubiquitination and NF-κB activation are impaired in the patient's fibroblasts stimulated by IL-1ß or TNF. In contrast, the patient's monocytes respond to IL-1ß more vigorously than control monocytes. However, the activation and differentiation of the patient's B cells are impaired in response to CD40 engagement. These cellular and clinical phenotypes largely overlap those of HOIL-1-deficient patients. Clinical differences between HOIL-1- and HOIP-mutated patients may result from differences between the mutations, the loci, or other factors. Our findings show that human HOIP is essential for the assembly and function of LUBAC and for various processes governing inflammation and immunity in both hematopoietic and nonhematopoietic cells.

Optineurin regulates the interferon response in a cell cycle-dependent manner.

Viral invasion into a host is initially recognized by the innate immune system, mainly through activation of the intracellular cytosolic signaling pathway and coordinated activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB) transcription factors that promote type I interferon gene induction. The TANK-binding Kinase 1 (TBK1) phosphorylates and activates IRF3. Here, we show that Optineurin (Optn) dampens the antiviral innate immune response by targeting the deubiquitinating enzyme CYLD to TBK1 in order to inhibit its enzymatic activity. Importantly, we found that this regulatory mechanism is abolished at the G2/M phase as a consequence of the nuclear translocation of CYLD and Optn. As a result, we observed, at this cell division stage, an increased activity and phosphorylation of TBK1 that lead to its relocalization to mitochondria and to enhanced interferon production, suggesting that this process, which relies on Optn function, might be of major importance to mount a preventive antiviral response during mitosis.

Phenotypic complementation of genetic immunodeficiency by chronic herpesvirus infection.

Variation in the presentation of hereditary immunodeficiencies may be explained by genetic or environmental factors. Patients with mutations in HOIL1 (RBCK1) present with amylopectinosis-associated myopathy with or without hyper-inflammation and immunodeficiency. We report that barrier-raised HOIL-1-deficient mice exhibit amylopectin-like deposits in the myocardium but show minimal signs of hyper-inflammation. However, they show immunodeficiency upon acute infection with Listeria monocytogenes, Toxoplasma gondii or Citrobacter rodentium. Increased susceptibility to Listeria was due to HOIL-1 function in hematopoietic cells and macrophages in production of protective cytokines. In contrast, HOIL-1-deficient mice showed enhanced control of chronic Mycobacterium tuberculosis or murine γ-herpesvirus 68 (MHV68), and these infections conferred a hyper-inflammatory phenotype. Surprisingly, chronic infection with MHV68 complemented the immunodeficiency of HOIL-1, IL-6, Caspase-1 and Caspase-1;Caspase-11-deficient mice following Listeria infection. Thus chronic herpesvirus infection generates signs of auto-inflammation and complements genetic immunodeficiency in mutant mice, highlighting the importance of accounting for the virome in genotype-phenotype studies.

TNF and IL-1 exhibit distinct ubiquitin requirements for inducing NEMO-IKK supramolecular structures.

Nuclear factor κB (NF-κB) essential modulator (NEMO), a regulatory component of the IκB kinase (IKK) complex, controls NF-κB activation through its interaction with ubiquitin chains. We show here that stimulation with interleukin-1 (IL-1) and TNF induces a rapid and transient recruitment of NEMO into punctate structures that are anchored at the cell periphery. These structures are enriched in activated IKK kinases and ubiquitinated NEMO molecules, which suggests that they serve as organizing centers for the activation of NF-κB. These NEMO-containing structures colocalize with activated TNF receptors but not with activated IL-1 receptors. We investigated the involvement of nondegradative ubiquitination in the formation of these structures, using cells deficient in K63 ubiquitin chains or linear ubiquitin chain assembly complex (LUBAC)-mediated linear ubiquitination. Our results indicate that, unlike TNF, IL-1 requires K63-linked and linear ubiquitin chains to recruit NEMO into higher-order complexes. Thus, different mechanisms are involved in the recruitment of NEMO into supramolecular complexes, which appear to be essential for NF-κB activation.

Plk1-dependent phosphorylation of optineurin provides a negative feedback mechanism for mitotic progression.

Plk1 activation is required for progression through mitotic entry to cytokinesis. Here we show that at mitotic entry, Plk1 phosphorylates Optineurin (Optn) at serine 177 and that this dissociates Optn from the Golgi-localized GTPase Rab8, inducing its translocation into the nucleus. Mass spectrometry analysis revealed that Optn is associated with a myosin phosphatase complex (MP), which antagonizes the mitotic function of Plk1. Our data also indicate that Optn functionally connects this complex to Plk1 by promoting phosphorylation of the myosin phosphatase targeting subunit 1 (MYPT1). Accordingly, silencing Optn expression increases Plk1 activity and induces abscission failure and multinucleation, which were rescued upon expression of wild-type (WT) Optn, but not a phospho-deficient mutant (S177A) that cannot translocate into the nucleus during mitosis. Overall, these results highlight an important role of Optn in the spatial and temporal coordination of Plk1 activity.

PP2A-mediated dephosphorylation of p107 plays a critical role in chondrocyte cell cycle arrest by FGF.

FGF signaling inhibits chondrocyte proliferation, a cell type-specific response that is the basis for several genetic skeletal disorders caused by activating FGFR mutations. This phenomenon requires the function of the p107 and p130 members of the Rb protein family, and p107 dephosphorylation is one of the earliest distinguishing events in FGF-induced growth arrest. To determine whether p107 dephoshorylation played a critical role in the chondrocyte response to FGF, we sought to counteract this process by overexpressing in RCS chondrocytes the cyclin D1/cdk4 kinase complex. CyclinD/cdk4-expressing RCS cells became resistant to FGF-induced p107 dephosphorylation and growth arrest, and maintained significantly high levels of cyclin E/cdk2 activity and of phosphorylated p130 at later times of FGF treatment. We explored the involvement of a phosphatase in p107 dephosphorylation. Expression of the SV40 small T-Ag, which inhibits the activity of the PP2A phosphatase, or knockdown of the expression of the PP2A catalytic subunit by RNA interference prevented p107 dephosphorylation and FGF-induced growth arrest of RCS cells. Furthermore, an association between p107 and PP2A was induced by FGF treatment. Our data show that p107 dephosphorylation is a key event in FGF-induced cell cycle arrest and indicate that in chondrocytes FGF activates the PP2A phosphatase to promote p107 dephosphorylation.

Activation of the ERK1/2 and p38 mitogen-activated protein kinase pathways mediates fibroblast growth factor-induced growth arrest of chondrocytes.

Fibroblast growth factors (FGFs) regulate long bone development by affecting the proliferation and differentiation of chondrocytes. FGF treatment inhibits the proliferation of chondrocytes both in vitro and in vivo, but the signaling pathways involved have not been clearly identified. In this report we show that both the MEK-ERK1/2 and p38 MAPK pathways, but not phospholipase C gamma or phosphatidylinositol 3-kinase, play a role in FGF-mediated growth arrest of chondrocytes. Chemical inhibitors of the MEK1/2 or the p38 MAPK pathways applied to rat chondrosarcoma (RCS) chondrocytes significantly prevented FGF-induced growth arrest. The retinoblastoma family members p107 and p130 were previously shown to be essential effectors of FGF-induced growth arrest in chondrocytes. The dephosphorylation of p107, one of the earliest events in RCS growth arrest, was significantly blocked by MEK1/2 inhibitors but not by the p38 MAPK inhibitors, whereas that of p130, which occurs later, was partially prevented both by the MEK and p38 inhibitors. Furthermore, by expressing the nerve growth factor (NGF) receptor, TrkA, and the epidermal growth factor (EGF) receptor, ErbB1, in RCS cells we show that NGF treatment of the transfected cells caused growth inhibition, whereas EGF did not. FGF- and NGF-induced growth inhibition is accompanied by a strong and sustained activation of ERK1/2 and p38 MAPK and a decrease of AKT phosphorylation, whereas EGF induces a much more transient activation of p38 and ERK1/2 and increases AKT phosphorylation. These results indicate that inhibition of chondrocyte proliferation by FGF requires both ERK1/2 and p38 MAPK signaling and also suggest that sustained activation of these pathways is required to achieve growth inhibition.

A network of transcriptional and signaling events is activated by FGF to induce chondrocyte growth arrest and differentiation.

Activating mutations in FGF receptor 3 (FGFR3) cause several human dwarfism syndromes by affecting both chondrocyte proliferation and differentiation. Using microarray and biochemical analyses of FGF-treated rat chondrosarcoma chondrocytes, we show that FGF inhibits chondrocyte proliferation by initiating multiple pathways that result in the induction of antiproliferative functions and the down-regulation of growth-promoting molecules. The initiation of growth arrest is characterized by the rapid dephosphorylation of the retinoblastoma protein (pRb) p107 and repression of a subset of E2F target genes by a mechanism that is independent of cyclin E-Cdk inhibition. In contrast, hypophosphorylation of pRb and p130 occur after growth arrest is first detected, and may contribute to its maintenance. Importantly, we also find a number of gene expression changes indicating that FGF promotes many aspects of hypertrophic differentiation, a notion supported by in situ analysis of developing growth plates from mice expressing an activated form of FGFR3. Thus, FGF may coordinate the onset of differentiation with chondrocyte growth arrest in the developing growth plate.

FGF signaling targets the pRb-related p107 and p130 proteins to induce chondrocyte growth arrest.

Unregulated FGF signaling affects endochondral ossification and long bone growth, causing several genetic forms of human dwarfism. One major mechanism by which FGFs regulate endochondral bone growth is through their inhibitory effect on chondrocyte proliferation. Because mice with targeted mutations of the retinoblastoma (Rb)-related proteins p107 and p130 present severe endochondral bone defects with excessive chondrocyte proliferation, we have investigated the role of the Rb family of cell cycle regulators in the FGF response. Using a chondrocyte cell line, we found that FGF induced a rapid dephosphorylation of all three proteins of the Rb family (pRb, p107, and p130) and a blockade of the cells in the G1 phase of the cell cycle. This cell cycle block was reversed by inactivation of Rb proteins with viral oncoproteins such as polyoma large T (PyLT) antigen and Adenovirus E1A. Expression of a PyLT mutant that efficiently binds pRb, but not p107 and p130, allowed the cells to be growth inhibited by FGF, suggesting that pRb itself is not involved in the FGF response. To investigate more precisely the role of the individual Rb family proteins in FGF-mediated growth inhibition, we used chondrocyte micromass culture of limb bud cells isolated from mice lacking Rb proteins individually or in combination. Although wild-type as well as Rb-/- chondrocytes were similarly growth inhibited by FGF, chondrocytes null for p107 and p130 did not respond to FGF. Furthermore, FGF treatment of metatarsal bone rudiments obtained from p107-/-;p130-/- embryos failed to inhibit proliferation of growth plate chondrocytes, whereas rudiments from p107-null or p130-null embryos showed only a slight inhibition of growth. Our findings indicate that p107 and p130, but not pRb, are critical effectors of FGF-mediated growth inhibition in chondrocytes.

The PDZ domain of TIP-2/GIPC interacts with the C-terminus of the integrin alpha5 and alpha6 subunits.

Different cDNA libraries were screened by the yeast two-hybrid system using as a bait the cytoplasmic sequence of integrin alpha6A or alpha6B subunits. Surprisingly, the same PDZ domain-containing protein, TIP-2/GIPC, was isolated with either of the variants, although their sequences are different. Direct interaction assays with the cytoplasmic domain of the integrin alpha1--7 subunits revealed that in addition to alpha6A and alpha6B, TIP-2/GIPC reacted also with alpha5, but not other alpha integrin subunits. The specificity of the interaction was confirmed by in vitro protein binding assays with purified peptides corresponding to integrin cytoplasmic domains. Further analysis with either truncation fragments of TIP-2/GIPC or mutated integrin cytoplasmic domains indicated that the interaction occurs between the PDZ domain of TIP-2/GIPC and a consensus PDZ domain-binding sequence, SDA, present at the C-terminus of the integrin alpha5 and alpha6A subunits. The integrin alpha6B subunit terminates with a different sequence, SYS, which may represent a new PDZ domain-binding motif.