The nuclease activity of DNA2 promotes exonuclease 1–independent mismatch repair

The DNA mismatch repair (MMR) system is a major DNA repair system that corrects DNA replication errors. In eukaryotes, the MMR system functions via mechanisms both dependent on and independent of exonuclease 1 (EXO1), an enzyme that has multiple roles in DNA metabolism. Although the mechanism of EXO1-dependent MMR is well understood, less is known about EXO1-independent MMR. Here, we provide genetic and biochemical evidence that the DNA2 nuclease/helicase has a role in EXO1-independent MMR. Biochemical reactions reconstituted with purified human proteins demonstrated that the nuclease activity of DNA2 promotes an EXO1-independent MMR reaction via a mismatch excision-independent mechanism that involves DNA polymerase δ. We show that DNA polymerase ε is not able to replace DNA polymerase δ in the DNA2-promoted MMR reaction. Unlike its nuclease activity, the helicase activity of DNA2 is dispensable for the ability of the protein to enhance the MMR reaction. Further examination established that DNA2 acts in the EXO1-independent MMR reaction by increasing the strand-displacement activity of DNA polymerase δ. These data reveal a mechanism for EXO1-independent mismatch repair.

The mismatch repair (MMR) system has been conserved from bacteria to humans (1, 2). It promotes genome stability by suppressing spontaneous and DNA damage-induced mutations (1, 3, 4, 5, 6, 7, 8, 9, 10, 11). The key function of the MMR system is the correction of DNA replication errors that escape the proofreading activities of replicative DNA polymerases (1, 4, 5, 6, 7, 8, 9, 10, 12). In addition, the MMR system removes mismatches formed during strand exchange in homologous recombination, suppresses homeologous recombination, initiates apoptosis in response to irreparable DNA damage caused by several anticancer drugs, and contributes to instability of triplet repeats and alternative DNA structures (1, 4, 5, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18). The principal components of the eukaryotic MMR system are MutSα (MSH2-MSH6 heterodimer), MutLα (MLH1-PMS2 heterodimer in humans and Mlh1-Pms1 heterodimer in yeast), MutSβ (MSH2-MSH3 heterodimer), proliferating cell nuclear antigen (PCNA), replication factor C (RFC), exonuclease 1 (EXO1), RPA, and DNA polymerase δ (Pol δ). Loss-of-function mutations in the MSH2, MLH1, MSH6, and PMS2 genes of the human MMR system cause Lynch and Turcot syndromes, and hypermethylation of the MLH1 promoter is responsible for ∼15% of sporadic cancers in several organs (19, 20). MMR deficiency leads to cancer initiation and progression via a multistage process that involves the inactivation of tumor suppressor genes and action of oncogenes (21).MMR occurs behind the replication fork (22, 23) and is a major determinant of the replication fidelity (24). The correction of DNA replication errors by the MMR system increases the replication fidelity by ∼100 fold (25). Strand breaks in leading and lagging strands as well as ribonucleotides in leading strands serve as signals that direct the eukaryotic MMR system to remove DNA replication errors (26, 27, 28, 29, 30). MMR is more efficient on the lagging than the leading strand (31). The substrates for MMR are all six base–base mismatches and 1 to 13-nt insertion/deletion loops (25, 32, 33, 34). Eukaryotic MMR commences with recognition of the mismatch by MutSα or MutSβ (32, 34, 35, 36). MutSα is the primary mismatch-recognition factor that recognizes both base–base mismatches and small insertion/deletion loops whereas MutSβ recognizes small insertion/deletion loops (32, 34, 35, 36, 37). After recognizing the mismatch, MutSα or MutSβ cooperates with RFC-loaded PCNA to activate MutLα endonuclease (38, 39, 40, 41, 42, 43). The activated MutLα endonuclease incises the discontinuous daughter strand 5′ and 3′ to the mismatch. A 5'' strand break formed by MutLα endonuclease is utilized by EXO1 to enter the DNA and excise a discontinuous strand portion encompassing the mismatch in a 5''→3′ excision reaction stimulated by MutSα/MutSβ (38, 44, 45). The generated gap is filled in by the Pol δ holoenzyme, and the nick is ligated by a DNA ligase (44, 46, 47). DNA polymerase ε (Pol ε) can substitute for Pol δ in the EXO1-dependent MMR reaction, but its activity in this reaction is much lower than that of Pol δ (48). Although MutLα endonuclease is essential for MMR in vivo, 5′ nick-dependent MMR reactions reconstituted in the presence of EXO1 are MutLα-independent (44, 47, 49).EXO1 deficiency in humans does not seem to cause significant cancer predisposition (19). Nevertheless, it is known that Exo1^-/- mice are susceptible to the development of lymphomas (50). Genetic studies in yeast and mice demonstrated that EXO1 inactivation causes only a modest defect in MMR (50, 51, 52, 53). In agreement with these genetic studies, a defined human EXO1-independent MMR reaction that depends on the strand-displacement DNA synthesis activity of Pol δ holoenzyme to remove the mismatch was reconstituted (54). Furthermore, an EXO1-independent MMR reaction that occurred in a mammalian cell extract system without the formation of a gapped excision intermediate was observed (54). Together, these findings implicated the strand-displacement activity of Pol δ holoenzyme in EXO1-independent MMR.In this study, we investigated DNA2 in the context of MMR. DNA2 is an essential multifunctional protein that has nuclease, ATPase, and 5''→3′ helicase activities (55, 56, 57). Previous research ascertained that DNA2 removes long flaps during Okazaki fragment maturation (58, 59, 60), participates in the resection step of double-strand break repair (61, 62, 63), initiates the replication checkpoint (64), and suppresses the expansions of GAA repeats (65). We have found in vivo and in vitro evidence that DNA2 promotes EXO1-independent MMR. Our data have indicated that the nuclease activity of DNA2 enhances the strand-displacement activity of Pol δ holoenzyme in an EXO1-independent MMR reaction.     

IFT trains overcome an NPHP module barrier at the transition zone

Cilia harbor diffusion barriers for soluble and membrane proteins within their proximal-most transition zone (TZ) region and employ an intraflagellar transport (IFT) system to form dynamic motile and signaling compartments. In this issue, De-Castro and colleagues (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202010178) uncover a long-suspected role for the TZ in gating IFT particles.

The cilium is a complex and functionally versatile cellular extension that emerged, some two billion years ago, in the lineage leading to the last eukaryotic common ancestor. To this day, motile cilia continue to enable locomotion in most unicellular eukaryotes and power sperm movement or fluid flow in metazoans. The sensory functions of motile cilia have also been adopted by and expanded in different metazoan cell types, to create specialized nonmotile cellular antennae (1).The formation and functions of cilia depend on a basal body from which stems the microtubule-based axoneme, as well as two additional, evolutionarily conserved macromolecular complexes: a transition zone (TZ) “ciliary gate” and an intraflagellar transport (IFT) machinery (Fig. 1). Understanding the functions and potential interactions between these ancient complexes is important, as they are involved in multiple human disorders—ciliopathies—that affect virtually all organ systems (1).Open in a separate windowFigure 1.The ciliary TZ acts as a barrier that must be overcome by the IFT system. TZ modules are known to assemble into diffusion barriers for soluble and membrane proteins at the base of cilia. De-Castro et al. (9) uncover a TZ barrier for the ciliary cargo-trafficking IFT system, which consists of different modules (BBSome, IFT-A, IFT-B) moved bidirectionally by kinesin anterograde and dynein-2 retrograde motors. When the dynein-2 subunit WDR-60 is disrupted, fewer dynein-2 retrograde motors associate with IFT particles upon entering cilia, and the under-powered retrograde IFT trains fail to break through the TZ—that is, unless the entire TZ is disrupted (MKS-5 mutant) or a specific TZ module (NPHP) is removed.The TZ, comprising over one dozen components, is characterized by Y-link structures that connect the axoneme to the membrane at the ciliary base. Studies in model systems, including Chlamydomonas, Caenorhabditis elegans, and mammals, establish the TZ as a diffusion barrier for membrane-associated proteins (2, 3). Mechanistically, how the TZ achieves this is unclear. One possibility is that membrane-associated TZ proteins create a lipid microdomain that limits the diffusion of membrane proteins (2, 4). The TZ also creates a separate barrier for soluble proteins. This gate, or ciliary pore complex, has the properties of a size-selective matrix that may share components and functional similarities with the nuclear pore complex (4, 5).How the different TZ proteins assemble in the context of Y-links to create these two distinct diffusion barriers remains unclear. Protein–protein interaction studies and genetic analyses (6) point to the existence of two multi-protein modules, termed MKS (Meckel–Gruber syndrome) and NPHP (nephronophthisis; Fig. 1). The modules are anchored at the TZ by at least two scaffolding proteins needed for the formation of Y-links. CEP290 tethers the MKS module, and MKS5 (RPGRIP1L) plays a more central role, assembling CEP290, the MKS module, and the NPHP module at the TZ (7). The MKS and NPHP modules are similarly required to establish the membrane and soluble protein gates; hence, their individual roles have been difficult to ascertain.The IFT machinery harbors ∼50 proteins and forms “trains” with relatively well-understood roles in shuttling ciliary cargo (8). IFT particles dock at basal body-associated transition fibers, travel to the tip using IFT-kinesin anterograde motors, and after remodeling, return to the base via an IFT–dynein (dynein-2) retrograde motor complex (Fig. 1). IFT trains continuously transit the TZ, motoring along doublet microtubules, contacting the overlying membrane, and passing in between Y-links (4). Evidence for physical and genetic interactions between IFT and TZ components suggests functional connections between them (6), but the obvious question of whether the TZ acts as a gate for IFT particles/trains remained largely unexplored. Until now.To understand the role of the C. elegans dynein-2 subunit WDR-60 (DYNC2I1/WDR60) in retrograde IFT, De-Castro and colleagues (9) analyzed two wdr-60 mutants, one null, and another lacking a β-propeller domain known to bind the IFT-dynein heavy chain (CHE-3; human DYNC2H1 orthologue). They found that loss of WDR-60 appears well-tolerated compared with that of CHE-3 or the light intermediate chain XBX-1 (human DYNC2LI1 orthologue). Ciliary structures in WDR-60–deficient animals appear normal; in contrast, like in other organisms, CHE-3 and XBX-1 are essential for retrograde IFT, and their disruption leads to short, bulbous structures.Yet, closer inspection of fluorescently labeled IFT reporters in wdr-60 mutants by live imaging revealed a significant defect: fewer IFT–dynein motors were incorporated onto anterograde IFT rafts and entered cilia (Fig. 1; 9). This in itself was not unexpected, as the Stephens and Nakayama laboratories had observed less DYNC2LI1 localized to cilia upon disrupting mammalian WDR-60 (10, 11). However, whereas mammalian cilia displayed strong IFT protein accumulations, particularly at the ciliary tip, C. elegans IFT particles lacking WDR-60 accumulated substantially less at the tip and were better able to traffic toward the base. Remarkably, though, the WDR-60–deficient IFT trains, with fewer IFT–dynein motors, amassed at the distal end of the TZ, apparently unable to cross the barrier (Fig. 1; 9). This finding presented an opportunity to further investigate how the TZ establishes a barrier for the IFT system.Confirming that the TZ acts like a gate was straightforward: The IFT roadblock was cleared in the mks-5 mutant, which completely lacks Y-links and the MKS and NPHP modules (Fig. 1). To narrow down which TZ module(s) provide the IFT-gating function, the researchers disrupted the MKS module, which contains many membrane-associated proteins and thus likely contributes to forming a membrane diffusion barrier. This did not restore the ability of WDR-60–deficient retrograde IFT trains to cross the TZ barrier. Similarly, the barrier remained intact upon mutation of CEP290, the anchor for the MKS module (Fig. 1). This left open the possibility that the NPHP module harbors the gating functionality.That is exactly what De-Castro et al. observed. Even though MKS-5, CEP-290, the MKS module, and Y-links are still present upon disrupting the NPHP module (nphp-4 mutant), IFT trains devoid of WDR-60 were now able to cross the TZ barrier (Fig. 1). Additionally, the nphp-4 mutant displayed faster anterograde and retrograde IFT speeds within the TZ compared with wild-type, further implying a role for the NPHP module in restricting IFT particle movement.Altogether, the findings by De-Castro et al. reveal that IFT trains driven by retrograde dynein motors must overcome, or “power through,” a TZ barrier specifically established by the NPHP module. But what exactly is the nature of this barrier? Is there some sort of molecular switch, the equivalent of a cell-cycle checkpoint where permission for entry and/or exit is regulated? Is it the purported gel-like matrix formed by nucleoporins (5) that simply decelerates the IFT trains? This latter hypothesis was not investigated or suggested in the current study. However, using mammalian cells, the Verhey laboratory (12) showed a physical interaction between a nucleoporin (NUP62) and the same TZ protein (NPHP4) found to confer the IFT-barrier functionality in C. elegans. This could in principle explain the findings of De-Castro et al.: removal of NPHP4 could prevent the proper localization of a related nucleoporin and disrupt the size-selective matrix at or near the TZ. Interestingly, the Verhey laboratory also revealed that NUP62 is required for ciliary entry of KIF17 (13), an IFT-associated kinesin motor (8).Connecting the proverbial dots from different model systems might be premature. But if correct, this hypothesis suggests that during the evolution of the ancestral eukaryote, the use of a nuclear pore complex–like system by the TZ to form a soluble protein diffusion barrier may have required specific adaptations for the ciliary entry/exit of the IFT system. Notably, that a ciliary pore complex could potentially influence ciliary entry and exit of IFT particles was suggested by Rosenbaum and Witman 20 yr ago (14). Continuing to shed light on the functional interactions between the TZ and IFT machinery will undoubtedly lead to a better understanding of how cilia create dynamic signaling compartments.     

New-Onset Diabetes Mellitus After Transplantation in a Cynomolgus Macaque (Macaca fasicularis)

A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal''s blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.Abbreviations: NODAT, new-onset diabetes mellitus after transplantationNew-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;^14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge.     

Spontaneous Osteoblastic Osteosarcoma in a Mongolian Gerbil (Meriones unguiculatus)

Spontaneous neoplasms in Mongolian gerbils have an incidence of 20% to 26.8%, but osteosarcomas occur at a much lower rate. Here we report a 1-y-old Mongolian gerbil with a spontaneous osteosarcoma at the level of the proximal tibia, with metastases to the pectoral muscles and lungs. Grossly, the tibial mass obliterated the tibia and adjacent muscles, and an axillary mass with a bloody, cavitary center expanded the pectoral muscles. Microscopically, the tibial mass was an infiltrative, osteoblastic mesenchymal neoplasm, and the axillary mass was an anaplastic mesenchymal neoplasm with hemorrhage. The lung contained multiple metastatic foci. Immunohistochemistry for osteonectin was strongly positive in the tibial, axillary, and pulmonary metastases. Although osteosarcoma is the most common primary malignant bone neoplasm that occurs spontaneously in all laboratory and domestic animal species and humans, it arises less frequently than does other neoplasms. The current case of spontaneous osteoblastic osteosarcoma of the proximal tibia and metastases to the pectoral muscles and lung in a Mongolian gerbil is similar in presentation, histology, and predilection site of both osteoblastic and telangiectatic osteosarcomas in humans. In addition, this case is an unusual manifestation of osteosarcoma in the appendicular skeleton of a Mongolian gerbil.Mongolian gerbils are used frequently in biologic research,^{1,2,4,9,10,12-14} particularly in oncogenic studies and filariasis research studying Brugia malayi.² There have been several reports^{1,6,10,11,13-15} of spontaneous neoplasms, particularly in gerbils 2 y of age and older, typically occurring with the highest incidences in the skin, reproductive tract, and adrenal glands; however, neoplasms have also been reported in the thyroid, thymus, liver, kidney, pancreas, and bone.^{1,6,10,11,13-15} The incidence of spontaneous neoplasms occurring in the subfamily Gerbillinae ranges from 20% to 26.8%,^{1,6,10,11,13-15} depending on the study, age, and sex of the animals.With a lower incidence than those reported for other neoplasms, osteosarcomas in gerbils have been described in the ramus of the mandible and as an extraskeletal mass throughout the peritoneum.^10,11 The usual age of onset for osteosarcomas in Mongolian gerbils is approximately 3 y (36 to 39 mo); however, no tumor type has been reported at less than 2 y of age in this species.^10,11 Here we report a spontaneous osteosarcoma that occurred at the level of the proximal tibia, with metastases to the pectoral muscles and lung, in a 1-y-old Mongolian gerbil.     

Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.Neuropeptides are the largest and most diverse family of neurotransmitters. They are released from axon terminals and dendrites, diffuse to pre- or postsynaptic neuronal structures and activate membrane G-protein-coupled receptors. Prodynorphin (PDYN)-derived opioid peptides including dynorphin A (Dyn A), dynorphin B (Dyn B) and big dynorphin (Big Dyn) consisting of Dyn A and Dyn B are endogenous ligands for the κ-opioid receptor. Acting through this receptor, dynorphins regulate processing of pain and emotions, memory acquisition and modulate reward induced by addictive substances.^{1, 2, 3, 4} Furthermore, dynorphins may produce robust cellular and behavioral effects that are not mediated through opioid receptors.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29} As evident from pharmacological, morphological, genetic and human neuropathological studies, these effects are generally pathological, including cell death, neurodegeneration, neurological dysfunctions and chronic pain. Big Dyn is the most active pathogenic peptide, which is about 10- to 100-fold more potent than Dyn A, whereas Dyn B does not produce non-opioid effects.^{16, 17, 22, 25} Big Dyn enhances activity of acid-sensing ion channel-1a (ASIC1a) and potentiates ASIC1a-mediated cell death in nanomolar concentrations^{30, 31} and, when administered intrathecally, induces characteristic nociceptive behavior at femtomolar doses.^{17, 22} Inhibition of endogenous Big Dyn degradation results in pathological pain, whereas prodynorphin (Pdyn) knockout mice do not maintain neuropathic pain.^{22, 32} Big Dyn differs from its constituents Dyn A and Dyn B in its unique pattern of non-opioid memory-enhancing, locomotor- and anxiolytic-like effects.²⁵Pathological role of dynorphins is emphasized by the identification of PDYN missense mutations that cause profound neurodegeneration in the human brain underlying the SCA23 (spinocerebellar ataxia type 23), a very rare dominantly inherited neurodegenerative disorder.^{27, 33} Most PDYN mutations are located in the Big Dyn domain, demonstrating its critical role in neurodegeneration. PDYN mutations result in marked elevation in dynorphin levels and increase in its pathogenic non-opioid activity.^{27, 34} Dominant-negative pathogenic effects of dynorphins are not produced through opioid receptors.ASIC1a, glutamate NMDA (N-methyl-d-aspartate) and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate ion channels, and melanocortin and bradykinin B2 receptors have all been implicated as non-opioid dynorphin targets.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 30, 31, 35, 36} Multiplicity of these targets and their association with the cellular membrane suggest that their activation is a secondary event triggered by a primary interaction of dynorphins with the membrane. Dynorphins are among the most basic neuropeptides.^{37, 38} The basic nature is also a general property of anti-microbial peptides (AMPs) and amyloid peptides that act by inducing membrane perturbations, altering membrane curvature and causing pore formation that disrupts membrane-associated processes including ion fluxes across the membrane.³⁹ The similarity between dynorphins and these two peptide groups in overall charge and size suggests a similar mode of their interactions with membranes.In this study, we dissect the interactions of dynorphins with the cell membrane, the primary event in their non-receptor actions. Using fluorescence imaging, correlation spectroscopy and patch-clamp techniques, we demonstrate that dynorphin peptides accumulate in the plasma membrane in live cells and cause a profound transient increase in cell membrane conductance. Membrane poration by endogenous neuropeptides may represent a novel mechanism of signal transduction in the brain. This mechanism may underlie effects of dynorphins under pathological conditions including chronic pain and tissue injury.     

NPHP proteins: gatekeepers of the ciliary compartment

The cilia and the cytoplasm are separated by a region called the transition zone, where wedge-shaped structures link the microtubule doublets of the axoneme to the ciliary membrane, thereby forming a ciliary “gate.” In this issue, Craige et al. (J. Cell Biol. doi:10.1083/jcb.201006105) demonstrate in Chlamydomonas reinhardtii that Nphp6/cep290, which is mutated in nephronophthisis (NPHP), is an integral component of these connectors and maintains the structural integrity of this gate.Cilia, tiny hairlike organelles that protrude from the cell surface, are located on almost all polarized cell types of the human body. Although the basic structures of different types of cilia are similar, they exert various tissue-specific functions during development, tissue morphogenesis, and homeostasis. Their prevalence and involvement in various cellular functions could explain why cilia-related disorders (ciliopathies) can affect many organ systems. Ciliopathies can either involve single organs, such as cystic kidney disease, or can occur as multisystemic disorders, such as Bardet Biedl syndrome and nephronophthisis (NPHP)-related disorders with phenotypically variable and overlapping disease manifestations (Badano et al., 2006; Fliegauf et al., 2007). Among syndromic forms of cystic kidney diseases, NPHP is the most common and complex disorder in childhood. NPHP comprises a genetically heterogenous group of renal cystic disorders with an autosomal recessive inheritance pattern. NPHP can cause end-stage renal disease in early infancy, childhood, and adolescence, as well as in adulthood, and can be associated with extra-renal disease manifestations such as ocular motor apraxia (Cogan syndrome), retinitis pigmentosa, Leber congenital amaurosis, coloboma of the optic nerve, cerebellar vermis aplasia (Joubert syndrome), liver fibrosis, cranioectodermal dysplasia, cone-shaped epiphyses, asphyxiating thoracic dysplasia (Jeune’s syndrome), Ellis-van Creveld syndrome, and, rarely, situs inversus (Omran and Ermisch-Omran, 2008). In addition, it has been shown that NPHP mutations can cause Meckel syndrome, a perinatal lethal disease characterized by congenital cystic kidney disease and encephalocele.Several genes responsible for NPHP have been identified (summarized in Omran and Ermisch-Omran, 2008), and many of the encoded proteins, such as NPHP1, NPHP2 (inversin), NPHP3, NPHP4 (nephroretinin), NPHP6, and NPHP8, have been found to interact with each other (Olbrich et al., 2003; Mollet et al., 2005; Delous et al., 2007; Bergmann et al., 2008). Although important mechanistic insights in the pathogenesis of NPHP have been established, such as perturbed Wnt signaling, the exact functional role of NPHP proteins still remained enigmatic (Simons et al., 2005; Bergmann et al., 2008). In this issue, Craige et al. shed new light in the function of NPHP6. They demonstrate that NPHP6 is a structural component of the champagne glass–shaped structures that link the microtubular doublets of the axoneme to the ciliary necklace, a distinct portion of the ciliary membrane first described almost 40 yr ago (Gilula and Satir, 1972) . Up to now, nothing was known about the protein composition of this unique structure at the ciliary base.The ciliary compartment including the ciliary membrane is equipped with a distinct composition of proteins, and the compartment border is located at the transition zone, where intraflagellar transport (IFT) particles are involved in active transport of cargoes from and to the ciliary compartment across the compartment border driven by two kinesin-2 family members: the heterotrimeric KIF3A–KIF3B–KAP complex and the homodimeric KIF17 motor (Fig. 1). Interestingly, several studies demonstrated that NPHP proteins sublocalize to the ciliary base of primary cilia (NPHP1, NPHP4, NPHP6, NPHP8, NPHP9, and NPHP11) as well as to the connecting cilium of the photoreceptor (NPHP1, NPHP5, and NPHP6), which is considered to be the orthologous structure of the transition zone (Olbrich et al., 2003; Mollet et al., 2005; Otto et al., 2005; Sayer et al., 2006; Delous et al., 2007; Bergmann et al., 2008; Otto et al., 2008; Valente et al., 2010). Detailed analyses of proteins such as NPHP1 revealed specific and exclusive localization at the transition zone (Fig. 1 A), which suggests a possible gatekeeper-like functional role of NPHP proteins at the ciliary compartment border to control delivery and exit of proteins to and from the cilium, respectively (Fliegauf et al., 2006). During ciliogenesis, NPHP1 becomes immediately recruited to the transition zone, which indicates that NPHP proteins may also be important for formation of this organelle. Interestingly, localization of these proteins to the transition zone has been evolutionary conserved and is also observed in Caenorhabditis elegans (Jauregui et al., 2008).Open in a separate windowFigure 1.NPHP proteins function at the ciliary gate (transition zone). (A) Localization of nephrocystin (red, NPHP1) at the transition zone is shown in murine (mIMCD3) immotile renal cilia (top), immotile canine renal MDCK cilia (middle), and motile human respiratory cilia (bottom). The ciliary axoneme is stained with antibodies targeting acetylated α-tubulin (green). Bars, 5 µm. (B) The triplet microtubule structure of the basal body is converted into the axonemal doublet structure at the transition zone of primary cilia. Proximal transition y-shaped fibers (red) connect each outer microtubule doublet to the membrane and mark the border at which IFT proteins start to shuffle cargoes to and from the ciliary compartment. The ciliary compartment, including the ciliary membrane, is therefore equipped with a distinct composition of proteins such as polycystin-2 and BBS proteins (i.e., BBS4), which differs from the cytoplasm and the apical plasma membrane. NPHP6/CEP290 as well as other NPHP proteins (e.g., NPHP1) localize at the transition zone and probably function as gatekeepers that control access and exit of proteins to and from the ciliary compartment, respectively.In this issue Craige et al. (2010) exploit the excellent genetic and biochemical tools available in Chlamydomonas reinhardtii to investigate the role of cep290/Nphp6 in the regulation of ciliary protein trafficking. Using immunoelectron microscopy, they show that cep290 localizes to the wedge-shaped structures that bridge and connect the flagellar membrane to the axonemal outer doublets within the transition zone. Further ultrastructural studies revealed defects of those structures in cep290 mutants, which indicates that cep290 is essential for integrity of the ciliary “gate” and an integral component of this poorly characterized structure. Detailed analyses of anterograde and retrograde IFT transport kinetics did not reveal gross alterations, which indicated that cep290 does not regulate IFT motor activity. Mass spectrometry analyses of flagella identified a complex pattern of abnormal protein composition. Biochemistry analyses of the flagella found increased amounts of IFT complex B proteins and BBS4, and decreased levels of the IFT complex A protein IFT139 as well as polycystin-2, which confirms that cep290 functions as a gatekeeper to control protein content of the flagella compartment. Alteration of polycystin-2 and BBS4 levels might even explain the complex clinical phenotype of cystic kidney disease and BBS-like findings present in children affected by CEP290/NPHP6 mutations (den Hollander et al., 2006; Sayer et al., 2006; Valente et al., 2006; Baala et al., 2007).Craige et al. (2010) also make some interesting observations that could be relevant to somatic gene therapy. Using dikaryon rescue studies, they show that cep290 is a dynamic protein that shuttles between the cytoplasm and the transition zone and that can incorporate into preassembled mutant transition zones and restore function. These results could be applied toward targeted gene therapy in NPHP-related diseases, such as Leber congenital amaurosis, a retinal degeneration disease in which cep290 is frequently mutated. Expression of CEP290 by gene therapy vectors in photoreceptors of patients could restore ciliary function.The cellular biological findings presented by Craige et al. (2010) are of major scientific interest because they open a new NPHP research field focusing on the ciliary compartment border. Future studies will address the roles of other interacting NPHP proteins for the integrity and/or function of the ciliary gate. Cell type–specific differences of the composition of the ciliary gate might account for the phenotypic differences observed in NPHP patients. Recent findings indicate similarities between the mechanisms regulating nuclear and ciliary import. Consistently, ciliary targeting of the IFT motor protein KIF17 has been shown to be regulated by a ciliary-cytoplasmic gradient of the small GTPase Ran, with high levels of GTP-bound Ran (RanGTP) in the cilium (Dishinger et al., 2010). Furthermore, KIF17 interacts with the nuclear import protein importin-β2 in a manner dependent on the ciliary localization signals and inhibited by RanGTP. Thus, the wedge-shaped fibers may function as the ciliary equivalent of the nuclear pore. Further work will shed light on the relationship between the different components of this interesting structure.     

p62/SQSTM1 upregulation constitutes a survival mechanism that occurs during granulocytic differentiation of acute myeloid leukemia cells

The p62/SQSTM1 adapter protein has an important role in the regulation of several key signaling pathways and helps transport ubiquitinated proteins to the autophagosomes and proteasome for degradation. Here, we investigate the regulation and roles of p62/SQSTM1 during acute myeloid leukemia (AML) cell maturation into granulocytes. Levels of p62/SQSTM1 mRNA and protein were both significantly increased during all-trans retinoic acid (ATRA)-induced differentiation of AML cells through a mechanism that depends on NF-κB activation. We show that this response constitutes a survival mechanism that prolongs the life span of mature AML cells and mitigates the effects of accumulation of aggregated proteins that occurs during granulocytic differentiation. Interestingly, ATRA-induced p62/SQSTM1 upregulation was impaired in maturation-resistant AML cells but was reactivated when differentiation was restored in these cells. Primary blast cells of AML patients and CD34⁺ progenitors exhibited significantly lower p62/SQSTM1 mRNA levels than did mature granulocytes from healthy donors. Our results demonstrate that p62/SQSTM1 expression is upregulated in mature compared with immature myeloid cells and reveal a pro-survival function of the NF-κB/SQSTM1 signaling axis during granulocytic differentiation of AML cells. These findings may help our understanding of neutrophil/granulocyte development and will guide the development of novel therapeutic strategies for refractory and relapsed AML patients with previous exposure to ATRA.p62 or sequestosome 1 (p62/SQSTM1) is a scaffold protein, implicated in a variety of biological processes including those that control cell death, inflammation, and metabolism.^{1, 2} Through its multi-domain structure, p62/SQSTM1 interacts specifically with key signaling proteins, including atypical PKC family members, NF-κB, and mTOR to control cellular responses.^{3, 4, 5, 6, 7} p62/SQSTM1 functions also as a key mediator of autophagy. Through its interaction with LC3, an essential protein involved in autophagy, p62/SQSTM1 selectively directs ubiquitinated substrates to autophagosomes leading to their subsequent degradation in lysosomes.^{8, 9} At the molecular level, p62/SQSTM1 acts as a pro-tumoral molecule by ensuring efficient and selective activation of cell signaling axes involved in cell survival, proliferation, and metabolism (i.e., NF-κB, mTOR, and Nrf-2 pathways).^{3, 5, 6, 7, 10, 11, 12, 13} p62/SQSTM1 can also signal anti-tumoral responses either by inactivating the pro-oncogenic signaling through BCR-ABL¹⁴ and Wnt pathways^{15, 16} or by inducing the activation of caspase 8, a pro-death protein.^{17, 18} Interestingly, in response to stress, autophagy promotes the degradation of p62, thus limits the activation of p62-regulatory pathways that control tumorigenesis.¹⁰ In addition, p62/SQSTM1 controls pathways that modulate differentiation of normal and cancerous cells. For example, p62/SQSTM1 has been shown to antagonize basal ERK activity and adipocyte differentiation.¹⁹ In contrast, p62/SQSTM1 favors differentiation of osteoclasts,²⁰ osteoblasts,²¹ neurons,²² megakaryocytes²³ and macrophages.²⁴ The role and regulation of p62/SQSTM1 during leukemia cell differentiation has been poorly documented.Acute myeloid leukemia (AML) is a hematological disease characterized by multiple deregulated pathways resulting in a blockade of myeloid precursors at different stages of maturation.^{25, 26} Acute promyelocyte leukemia (APL) is the M3 type of AML characterized by an arrest of the terminal differentiation of promyelocytes into granulocytes and frequently associated with the expression of the oncogenic PML-RAR alpha fusion gene.^{27, 28} All-trans retinoic acid (ATRA), a potent activator of cellular growth arrest, differentiation, and death of APL cells, has been shown to effectively promote complete clinical remission of APL when combined with chemotherapy.^{29, 30, 31} Despite the success of this treatment, some APL patients are refractory to ATRA treatment or relapse owing to the development of resistance to ATRA in leukemia cells.^{32, 33, 34}Our previous results revealed that autophagy flux is activated during granulocyte differentiation of myeloid leukemia cell lines induced by ATRA.³⁵ In the present study, we observed that p62/SQSTM1, an autophagic substrate, is markedly upregulated at both mRNA and protein levels during the granulocytic differentiation process. Here, we investigated the regulation and the function of p62/SQSTM1 during AML cells differentiation into neutrophils/granulocytes.     

Induction of COX-2-PGE2 synthesis by activation of the MAPK/ERK pathway contributes to neuronal death triggered by TDP-43-depleted microglia

Neuroinflammation is a striking hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Previous studies have shown the contribution of glial cells such as astrocytes in TDP-43-linked ALS. However, the role of microglia in TDP-43-mediated motor neuron degeneration remains poorly understood. In this study, we show that depletion of TDP-43 in microglia, but not in astrocytes, strikingly upregulates cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production through the activation of MAPK/ERK signaling and initiates neurotoxicity. Moreover, we find that administration of celecoxib, a specific COX-2 inhibitor, greatly diminishes the neurotoxicity triggered by TDP-43-depleted microglia. Taken together, our results reveal a previously unrecognized non-cell-autonomous mechanism in TDP-43-mediated neurodegeneration, identifying COX-2-PGE2 as the molecular events of microglia- but not astrocyte-initiated neurotoxicity and identifying celecoxib as a novel potential therapy for TDP-43-linked ALS and possibly other types of ALS.Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the degeneration of motor neurons in the brain and spinal cord.¹ Most cases of ALS are sporadic, but 10% are familial. Familial ALS cases are associated with mutations in genes such as Cu/Zn superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TARDBP) and, most recently discovered, C9orf72. Currently, most available information obtained from ALS research is based on the study of SOD1, but new studies focusing on TARDBP and C9orf72 have come to the forefront of ALS research.^{1, 2} The discovery of the central role of the protein TDP-43, encoded by TARDBP, in ALS was a breakthrough in ALS research.^{3, 4, 5} Although pathogenic mutations of TDP-43 are genetically rare, abnormal TDP-43 function is thought to be associated with the majority of ALS cases.¹ TDP-43 was identified as a key component of the ubiquitin-positive inclusions in most ALS patients and also in other neurodegenerative diseases such as frontotemporal lobar degeneration,^{6, 7} Alzheimer''s disease (AD)^{8, 9} and Parkinson''s disease (PD).^{10, 11} TDP-43 is a multifunctional RNA binding protein, and loss-of-function of TDP-43 has been increasingly recognized as a key contributor in TDP-43-mediated pathogenesis.^{5, 12, 13, 14}Neuroinflammation, a striking and common hallmark involved in many neurodegenerative diseases, including ALS, is characterized by extensive activation of glial cells including microglia, astrocytes and oligodendrocytes.^{15, 16} Although numerous studies have focused on the intrinsic properties of motor neurons in ALS, a large amount of evidence showed that glial cells, such as astrocytes and microglia, could have critical roles in SOD1-mediated motor neuron degeneration and ALS progression,^{17, 18, 19, 20, 21, 22} indicating the importance of non-cell-autonomous toxicity in SOD1-mediated ALS pathogenesis.Very interestingly, a vital insight of neuroinflammation research in ALS was generated by the evidence that both the mRNA and protein levels of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) are upregulated in both transgenic mouse models and in human postmortem brain and spinal cord.^{23, 24, 25, 26, 27, 28, 29} The role of COX-2 neurotoxicity in ALS and other neurodegenerative disorders has been well explored.^{30, 31, 32} One of the key downstream products of COX-2, prostaglandin E2 (PGE2), can directly mediate COX-2 neurotoxicity both in vitro and in vivo.^{33, 34, 35, 36, 37} The levels of COX-2 expression and PGE2 production are controlled by multiple cell signaling pathways, including the mitogen-activated protein kinase (MAPK)/ERK pathway,^{38, 39, 40} and they have been found to be increased in neurodegenerative diseases including AD, PD and ALS.^{25, 28, 32, 41, 42, 43, 44, 45, 46} Importantly, COX-2 inhibitors such as celecoxib exhibited significant neuroprotective effects and prolonged survival or delayed disease onset in a SOD1-ALS transgenic mouse model through the downregulation of PGE2 release.²⁸Most recent studies have tried to elucidate the role of glial cells in neurotoxicity using TDP-43-ALS models, which are considered to be helpful for better understanding the disease mechanisms.^{47, 48, 49, 50, 51} Although the contribution of glial cells to TDP-43-mediated motor neuron degeneration is now well supported, this model does not fully suggest an astrocyte-based non-cell autonomous mechanism. For example, recent studies have shown that TDP-43-mutant astrocytes do not affect the survival of motor neurons,^{50, 51} indicating a previously unrecognized non-cell autonomous TDP-43 proteinopathy that associates with cell types other than astrocytes.Given that the role of glial cell types other than astrocytes in TDP-43-mediated neuroinflammation is still not fully understood, we aim to compare the contribution of microglia and astrocytes to neurotoxicity in a TDP-43 loss-of-function model. Here, we show that TDP-43 has a dominant role in promoting COX-2-PGE2 production through the MAPK/ERK pathway in primary cultured microglia, but not in primary cultured astrocytes. Our study suggests that overproduction of PGE2 in microglia is a novel molecular mechanism underlying neurotoxicity in TDP-43-linked ALS. Moreover, our data identify celecoxib as a new potential effective treatment of TDP-43-linked ALS and possibly other types of ALS.     

Decreased mitochondrial priming determines chemoresistance of colon cancer stem cells

Tumor heterogeneity is in part determined by the existence of cancer stem cells (CSCs) and more differentiated tumor cells. CSCs are considered to be the tumorigenic root of cancers and suggested to be chemotherapy resistant. Here we exploited an assay that allowed us to measure chemotherapy-induced cell death in CSCs and differentiated tumor cells simultaneously. This confirmed that CSCs are selectively resistant to conventional chemotherapy, which we revealed is determined by decreased mitochondrial priming. In agreement, lowering the anti-apoptotic threshold using ABT-737 and WEHI-539 was sufficient to enhance chemotherapy efficacy, whereas ABT-199 failed to sensitize CSCs. Our data therefore point to a crucial role of BCLXL in protecting CSCs from chemotherapy and suggest that BH3 mimetics, in combination with chemotherapy, can be an efficient way to target chemotherapy-resistant CSCs.Colorectal cancer is the third most common cancer worldwide.^{1, 2} Patients with advanced stage colorectal cancer are routinely treated with 5-fluorouracil (5-FU), leucovorin and oxaliplatin (FOLFOX), or with 5-FU, leucovorin and irinotecan (FOLFIRI), often in combination with targeted agents such as anti-VEGF or anti-EGFR at metastatic disease.^{3, 4, 5, 6} Despite this intensive treatment, therapy is still insufficiently effective and chemotherapy resistance occurs frequently. Although still speculative, it has been suggested that unequal sensitivity to chemotherapy is due to an intratumoral heterogeneity that is orchestrated by cancer stem cells (CSCs) that can self-renew and give rise to more differentiated progeny.^{7, 8} When isolated from patients, CSCs efficiently form tumors upon xenotransplantation into mice which resemble the primary tumor from which they originated.^{9, 10, 11} In addition, many xenotransplantation studies have emphasized the importance of CSCs for tumor growth.^{9, 10, 11, 12} Colon CSCs were originally isolated from primary human colorectal tumor specimens using CD133 as cell surface marker and shown to be highly tumorigenic when compared with the non-CSCs population within a tumor.^{9, 10} Later, other cell surface markers as well as the activity of the Wnt pathway have been used to isolate colon CSCs from tumors.^{12, 13} Spheroid cultures have been established from human primary colorectal tumors that selectively enrich for the growth of colon CSCs,^{11, 12} although it is important to realize that these spheres also contain more differentiated tumor cells.¹² In agreement, we have shown that the Wnt activity reporter that directs the expression of enhanced green fluorescent protein (TOP-GFP) allows for the separation of CSCs from more differentiated progeny in the spheroid cultures.¹²CSCs are suggested to be responsible for tumor recurrence after initial therapy, as they are considered to be selectively resistant to therapy.^{11, 14} Conventional chemotherapy induces, among others, DNA damage and subsequent activation of the mitochondrial cell death pathway, which is regulated by a balance between pro- and anti-apoptotic BCL2 family members.¹⁵ Upon activation of apoptosis, pro-apoptotic BH3 molecules are activated and these may perturb the balance in favor of apoptosis initiated by mitochondrial outer membrane polarization (MOMP), release of cytochrome c and subsequent activation of a caspase cascade.The apoptotic balance of cancer cells can be measured with the use of a functional assay called BH3 profiling.¹⁶ BH3 profiling is a method to determine the apoptotic ‘priming'' level of a cell by exposing mitochondria to standardized amounts of roughly 20-mer peptides derived from the alpha-helical BH3 domains of BH3-only proteins and determining the rate of mitochondrial depolarization. Using this approach, priming was measured in various cancers and compared with normal tissues.^{17, 18} In all cancer types tested, the mitochondrial priming correlated well with the observed clinical response to chemotherapy. That is, cancers that are highly primed are more chemosensitive, whereas chemoresistant tumor cells and normal tissues are poorly primed.^{17, 18} This suggests that increasing mitochondrial priming can potentially increase chemosensitivity, which can be achieved by directly inhibiting the anti-apoptotic BCL2 family members.¹⁸ To this end, small-molecule inhibitors, so-called BH3 mimetics, have been developed. ABT-737 and the highly related ABT-263 both inhibit BCL2, BCLXL and BCLW^{19, 20, 21} and were shown to be effective in killing cancer cells in vitro and in vivo²¹ with a preference for BCL2.^{19, 22} As BCL2 protein expression is often upregulated in hematopoietic cancers, it represents a promising target, which was supported by high efficacy of these BH3 mimetics in animal experiments.²¹ However, in vivo efficacy is limited due to thrombocytopenia, which relates to a dependence of platelets on BCLXL for survival.^{23, 24} To overcome this toxicity, a BCL2-specific compound, ABT-199, was developed.²⁵ Souers et al.²⁵ showed that inhibition of BCL2 using ABT-199 blocks tumor growth of acute lymphoblastic leukemia cells in xenografts. In addition to the single compound effects of ABT-199, combination with rituximab inhibited growth of non-Hodgkin''s lymphoma, mantle cell lymphoma and acute lymphoblastic leukemia tumor cells growth in vivo.²⁵ Moreover, highly effective tumor lysis was observed in all three patients with chronic lymphocytic leukemia that were treated with ABT-199.²⁵ More recently, a BCLXL-specific compound, WEHI-539, was discovered using high-throughput chemical screening.²⁶As the apoptotic balance appears a useful target for the treatment of cancers and CSCs have been suggested to resist therapy selectively, we set out to analyze whether specifically colon CSCs are resistant to therapy and whether this is due to an enhanced anti-apoptotic threshold, specific to CSCs. To study chemosensitivity, we developed a robust single cell-based analysis in which we can measure apoptosis simultaneously in CSCs and their differentiated progeny. Utilizing this system we showed that colon CSCs and not their differentiated progeny are resistant to chemotherapeutic compounds and that this was due to the fact that these cells are less primed to mitochondrial death. Furthermore, inhibition of anti-apoptotic BCLXL molecule with either ABT-737 or WEHI-539, but not ABT-199, breaks this resistance and sensitizes the CSCs to chemotherapy.     

Necroptosis is preceded by nuclear translocation of the signaling proteins that induce it

A signaling pathway that induces programmed necrotic cell death (necroptosis) was reported to be activated in cells by several cytokines and various pathogen components. The major proteins participating in that pathway are the protein kinases RIPK1 and RIPK3 and the pseudokinase mixed lineage kinase domain-like protein (MLKL). Recent studies have suggested that MLKL, once activated, mediates necroptosis by binding to cellular membranes, thereby triggering ion fluxes. However, our knowledge of both the sequence of molecular events leading to MLKL activation and the subcellular sites of these events is fragmentary. Here we report that the association of MLKL with the cell membrane in necroptotic death is preceded by the translocation of phosphorylated MLKL, along with RIPK1 and RIPK3, to the nucleus.Apart from the apoptotic cell death pathway that ligands of the tumor necrosis factor (TNF) family can activate, these ligands and various other inducers, including the interferons and various pathogen components, have in recent years been found also to trigger a signaling cascade that induces programmed necrotic death (necroptosis). This cascade encompasses sequential activation of the protein kinases RIPK1 and RIPK3 and the pseudokinase mixed lineage kinase domain-like protein (MLKL).^{1, 2, 3, 4, 5} RIPK3-mediated phosphorylation of MLKL triggers its oligomerization, which is necessary and sufficient for the induction of cell death,^{6, 7, 8} and can also trigger some non-deadly functions.⁹ MLKL was recently suggested to trigger cell death by binding to cellular membranes and initiating ion fluxes through them.^{6, 7, 8, 10} However, its exact molecular target in death induction is contentious.^{6, 8, 10, 11, 12} Current knowledge of the subcellular sites of MLKL action is based mainly on determination of the location of this protein close to the time of cell death. Here we present a detailed assessment of the cellular location of MLKL at different times following its activation. Our findings indicate that before cell death, MLKL translocates to the nucleus along with RIPK1 and RIPK3.     

The Fcp1-Wee1-Cdk1 axis affects spindle assembly checkpoint robustness and sensitivity to antimicrotubule cancer drugs

To grant faithful chromosome segregation, the spindle assembly checkpoint (SAC) delays mitosis exit until mitotic spindle assembly. An exceedingly prolonged mitosis, however, promotes cell death and by this means antimicrotubule cancer drugs (AMCDs), that impair spindle assembly, are believed to kill cancer cells. Despite malformed spindles, cancer cells can, however, slip through SAC, exit mitosis prematurely and resist killing. We show here that the Fcp1 phosphatase and Wee1, the cyclin B-dependent kinase (cdk) 1 inhibitory kinase, play a role for this slippage/resistance mechanism. During AMCD-induced prolonged mitosis, Fcp1-dependent Wee1 reactivation lowered cdk1 activity, weakening SAC-dependent mitotic arrest and leading to mitosis exit and survival. Conversely, genetic or chemical Wee1 inhibition strengthened the SAC, further extended mitosis, reduced antiapoptotic protein Mcl-1 to a minimum and potentiated killing in several, AMCD-treated cancer cell lines and primary human adult lymphoblastic leukemia cells. Thus, the Fcp1-Wee1-Cdk1 (FWC) axis affects SAC robustness and AMCDs sensitivity.The spindle assembly checkpoint (SAC) delays mitosis exit to coordinate anaphase onset with spindle assembly. To this end, SAC inhibits the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) to prevent degradation of the anaphase inhibitor securin and cyclin B, the major mitotic cyclin B-dependent kinase 1 (cdk1) activator, until spindle assembly.¹ However, by yet poorly understood mechanisms, exceedingly prolonging mitosis translates into cell death induction.^{2, 3, 4, 5, 6, 7} Although mechanistic details are still missing on how activation of cell death pathways is linked to mitosis duration, prolongation of mitosis appears crucial for the ability of antimicrotubule cancer drugs (AMCDs) to kill cancer cells.^{2, 3, 4, 5, 6, 7} These drugs, targeting microtubules, impede mitotic spindle assembly and delay mitosis exit by chronically activating the SAC. Use of these drugs is limited, however, by toxicity and resistance. A major mechanism for resistance is believed to reside in the ability of cancer cells to slip through the SAC and exit mitosis prematurely despite malformed spindles, thus resisting killing by limiting mitosis duration.^{2, 3, 4, 5, 6, 7} Under the AMCD treatment, cells either die in mitosis or exit mitosis, slipping through the SAC, without or abnormally dividing.^{2, 3, 4} Cells that exit mitosis either die at later stages or survive and stop dividing or proliferate, giving rise to resistance.^{2, 3, 4} Apart from a role for p53, what dictates cell fate is still unknown; however, it appears that the longer mitosis is protracted, the higher the chances for cell death pathway activation are.^{2, 3, 4, 5, 6, 7}Although SAC is not required per se for killing,⁶ preventing SAC adaptation should improve the efficacy of AMCD by increasing mitosis duration.^{2, 3, 4, 5, 6, 7} Therefore, further understanding of the mechanisms by which cells override SAC may help to improve the current AMCD therapy. Several kinases are known to activate and sustain SAC, and cdk1 itself appears to be of primary relevance.^{1, 8, 9} By studying mitosis exit and SAC resolution, we recently reported a role for the Fcp1 phosphatase to bring about cdk1 inactivation.^{10, 11} Among Fcp1 targets, we identified cyclin degradation pathway components, such as Cdc20, an APC/C co-activator, USP44, a deubiquitinating enzyme, and Wee1.^{10, 11} Wee1 is a crucial kinase that controls the G2 phase by performing inhibitory phosphorylation of cdk1 at tyr-15 (Y15-cdk1). Wee1 is also in a feedback relationship with cdk1 itself that, in turn, can phosphorylate and inhibit Wee1 in an autoamplification loop to promote the G2-to-M phase transition.¹² At mitosis exit, Fcp1 dephosphorylated Wee1 at threonine 239, a cdk1-dependent inhibitory phosphorylation, to dampen down the cdk1 autoamplification loop, and Cdc20 and USP44, to promote APC/C-dependent cyclin B degradation.^{10, 11, 12} In this study we analysed the Fcp1 relevance in SAC adaptation and AMCD sensitivity.     

Omentin protects against LPS-induced ARDS through suppressing pulmonary inflammation and promoting endothelial barrier via an Akt/eNOS-dependent mechanism

Acute respiratory distress syndrome (ARDS) is characterized by increased pulmonary inflammation and endothelial barrier permeability. Omentin has been shown to benefit obesity-related systemic vascular diseases; however, its effects on ARDS are unknown. In the present study, the level of circulating omentin in patients with ARDS was assessed to appraise its clinical significance in ARDS. Mice were subjected to systemic administration of adenoviral vector expressing omentin (Ad-omentin) and one-shot treatment of recombinant human omentin (rh-omentin) to examine omentin''s effects on lipopolysaccharide (LPS)-induced ARDS. Pulmonary endothelial cells (ECs) were treated with rh-omentin to further investigate its underlying mechanism. We found that a decreased level of circulating omentin negatively correlated with white blood cells and procalcitonin in patients with ARDS. Ad-omentin protected against LPS-induced ARDS by alleviating the pulmonary inflammatory response and endothelial barrier injury in mice, accompanied by Akt/eNOS pathway activation. Treatment of pulmonary ECs with rh-omentin attenuated inflammatory response and restored adherens junctions (AJs), and cytoskeleton organization promoted endothelial barrier after LPS insult. Moreover, the omentin-mediated enhancement of EC survival and differentiation was blocked by the Akt/eNOS pathway inactivation. Therapeutic rh-omentin treatment also effectively protected against LPS-induced ARDS via the Akt/eNOS pathway. Collectively, these data indicated that omentin protects against LPS-induced ARDS by suppressing inflammation and promoting the pulmonary endothelial barrier, at least partially, through an Akt/eNOS-dependent mechanism. Therapeutic strategies aiming to restore omentin levels may be valuable for the prevention or treatment of ARDS.Acute respiratory distress syndrome (ARDS) is a devastating condition with a 30–60% mortality rate.^{1, 2} Although the pathogenesis of ARDS is complex, the inflammatory response and endothelial barrier disruption play important roles in the development of ARDS.^{3, 4, 5} Therefore, in addition to conventional anti-inflammatory treatments, therapeutic strategies aim to restore pulmonary endothelial barrier integrity and function through regulating inter-endothelial AJs and the endothelial cytoskeleton to minimize protein leakage and leukocyte infiltration under ARDS conditions.^{6, 7}Obesity, especially visceral obesity, has clearly been shown to impair systemic vasculature and to lead to the initiation and progression of vascular disorders.^{8, 9, 10} Although different from the well-documented impacts of obesity on cardiovascular disease, the relationships between obesity and ARDS have not been well elucidated. Clinical and experimental data focused on pertinent physiological changes in obesity indicate that the obesity may alter ARDS pathogenesis by ‘priming'' the pulmonary endothelial barrier for insult and amplifying the early inflammatory response, thus lowering the threshold to initiate ARDS.^{11, 12} Contrary to conventional dogma, adipose tissue is now appreciated as an important endocrine tissue that secretes various bioactive molecules called adipokines, which contribute to the progression of diverse vascular diseases, including hypertension, cardiovascular disease and atherosclerosis.^{13, 14, 15, 16} Although ARDS is not a classified pulmonary vascular disease, it is a severe inflammatory lung condition with widespread pulmonary endothelial breakdown. Clinical evidence has indicated that the obesity might be an emerging risk factor for ARDS and that circulating adipokines levels are associated with the initiation and progression of ARDS.^{11, 12, 17, 18} Moreover, experimental studies have suggested that some anti-inflammatory adipokines, such as adiponectin and apelin, exert beneficial actions on ARDS.^{19, 20, 21}Omentin is an anti-inflammatory adipokine that is abundant in human visceral fat tissue.^{22, 23} Paradoxically, higher circulating omentin-1 levels are present in lean and healthy individuals compared with the obese and diabetic patients. Moreover, as a novel biomarker of endothelial dysfunction, reduced circulating omentin levels are related to the pathological mechanism of obesity-linked vascular disorders, including type 2 diabetes, atherosclerosis, hypertension and cardiovascular disease.^{24, 25, 26, 27, 28} Furthermore, experimental studies have found that omentin stimulates vasodilation in isolated blood vessels and suppresses cytokine-stimulated inflammation in endothelial cells (ECs).^{29, 30, 31} Thus, these data suggest that omentin may protect against obesity-related vascular complications through its anti-inflammatory and vascular-protective properties; however, little is known regarding its role in lung tissue. It was reported that decreased circulating omentin-1 levels could be regarded as an independent predictive marker for the obstructive sleep apnea syndrome and that omentin protects against pulmonary arterial hypertension through inhibiting vascular structure remodeling and abnormal contractile reactivity.^{32, 33, 34} However, to our knowledge, no study has assessed the impact of omentin on ARDS.Akt-related signaling pathways function as an endogenous negative feedback mechanism in response to the injurious stimulus. Our prior studies have demonstrated that Akt-related signaling contributes to protection against ARDS.^{35, 36} Moreover, omentin has been reported to exert anti-inflammatory, pro-survival and pro-angiogenic functions in various cells via an Akt-dependent mechanism.^{30, 31, 37, 38, 39, 40, 41, 42}Collectively, given that ARDS is ultimately an obesity-related disorder of vascular function and that omentin is a favorable pleiotropic adipokine capable of anti-inflammatory, pro-angiogenic and anti-apoptotic abilities; omentin may exert beneficial effects on ARDS. In the present study, we first aimed to appraise the clinical significance of omentin in ARDS and then specifically evaluated its impact on inflammation and the endothelial barrier. Furthermore, we mechanistically investigated the role of Akt-related signaling pathways in these effects induced by omentin in vivo and in vitro.