Pharmacologic targeting of the p62 ZZ domain enhances both anti-tumor and bone-anabolic effects of bortezomib in multiple myeloma.

Multiple myeloma (MM) is a malignancy of plasma cells whose antibody secretion creates proteotoxic stress relieved by the N-end rule pathway, a proteolytic system that degrades Narginylated proteins in the proteasome. When the proteasome is inhibited, protein cargo is alternatively targeted for autophagic degradation by binding to the ZZ-domain of p62/sequestosome-1. Here, we demonstrate that XRK3F2, a selective ligand for the ZZ-domain, dramatically improved two major responses to the proteasome inhibitor bortezomib by increasing: 1) killing of human MM cells by stimulating both bortezomib mediated apoptosis and necroptosis, a process regulated by p62; and 2) preservation of bone mass by stimulating osteoblasts differentiation and inhibiting osteoclastic bone destruction. Co-administration of bortezomib and XRK3F2 inhibited both branches of the bimodal N-end rule pathway exhibited synergistic anti-MM effects on MM cell lines and CD138+ cells from MM patients, and prevented stromal-mediated MM cell survival. In mice with established human MM, coadministration of bortezomib and XRK3F2 decreased tumor burden and prevented the progression of MM-induced osteolytic disease by inducing new bone formation more effectively than either single agent alone. The results suggest that p62-ZZ ligands enhance the anti-MM efficacy of proteasome inhibitors and can reduce MM morbidity and mortality by improving bone health.

Pharmacological inhibition of Carbonic Anhydrase IX and XII to enhance targeting of acute myeloid leukaemia cells under hypoxic conditions.

Acute myeloid leukaemia (AML) is an aggressive form of blood cancer that carries a dismal prognosis. Several studies suggest that the poor outcome is due to a small fraction of leukaemic cells that elude treatment and survive in specialised, oxygen (O2 )-deprived niches of the bone marrow. Although several AML drug targets such as FLT3, IDH1/2 and CD33 have been established in recent years, survival rates remain unsatisfactory, which indicates that other, yet unrecognized, mechanisms influence the ability of AML cells to escape cell death and to proliferate in hypoxic environments. Our data illustrates that Carbonic Anhydrases IX and XII (CA IX/XII) are critical for leukaemic cell survival in the O2 -deprived milieu. CA IX and XII function as transmembrane proteins that mediate intracellular pH under low O2 conditions. Because maintaining a neutral pH represents a key survival mechanism for tumour cells in O2 -deprived settings, we sought to elucidate the role of dual CA IX/XII inhibition as a novel strategy to eliminate AML cells under hypoxic conditions. Our findings demonstrate that the dual CA IX/XII inhibitor FC531 may prove to be of value as an adjunct to chemotherapy for the treatment of AML.

Increased S1P expression in osteoclasts enhances bone formation in an animal model of Paget's disease.

Paget's disease (PD) is characterized by increased numbers of abnormal osteoclasts (OCLs) that drive exuberant bone formation, but the mechanisms responsible for the increased bone formation remain unclear. We previously reported that OCLs from 70% of PD patients express measles virus nucleocapsid protein (MVNP), and that transgenic mice with targeted expression of MVNP in OCLs (MVNP mice) develop bone lesions and abnormal OCLs characteristic of PD. In this report, we examined if OCL-derived sphingosine-1-phosphate (S1P) contributed to the abnormal bone formation in PD, since OCL-derived S1P can act as a coupling factor to increase normal bone formation via binding S1P-receptor-3 (S1PR3) on osteoblasts (OBs). We report that OCLs from MVNP mice and PD patients expressed high levels of sphingosine kinase-1 (SphK-1) compared with wild-type (WT) mouse and normal donor OCLs. SphK-1 production by MVNP-OCLs was interleukin-6 (IL-6)-dependent since OCLs from MVNP/IL-6-/- mice expressed lower levels of SphK-1. Immunohistochemistry of bone biopsies from a normal donor, a PD patient, WT and MVNP mice confirmed increased expression levels of SphK-1 in OCLs and S1PR3 in OBs of the PD patient and MVNP mice compared with normal donor and WT mice. Further, MVNP-OCLs cocultured with OBs from MVNP or WT mice increased OB-S1PR3 expression and enhanced expression of OB differentiation markers in MVNP-OBs precursors compared with WT-OBs, which was mediated by IL-6 and insulin-like growth factor 1 secreted by MVNP-OCLs. Finally, the addition of an S1PR3 antagonist (VPC23019) to WT or MVNP-OBs treated with WT and MVNP-OCL-conditioned media (CM) blocked enhanced OB differentiation of MVNP-OBs treated with MVNP-OCL-CM. In contrast, the addition of the SIPR3 agonist, VPC24191, to the cultures enhanced osterix and Col-1A expression in MVNP-OBs treated with MVNP-OCL-CM compared with WT-OBs treated with WT-OCL-CM. These results suggest that IL-6 produced by PD-OCLs increases S1P in OCLs and S1PR3 on OBs, to increase bone formation in PD.

Classic and targeted anti-leukaemic agents interfere with the cholesterol biogenesis metagene in acute myeloid leukaemia: Therapeutic implications.

Despite significant advances in deciphering the molecular landscape of acute myeloid leukaemia (AML), therapeutic outcomes of this haematological malignancy have only modestly improved over the past decades. Drug resistance and disease recurrence almost invariably occur, highlighting the need for a deeper understanding of these processes. While low O2 compartments, such as bone marrow (BM) niches, are well-recognized hosts of drug-resistant leukaemic cells, standard in vitro studies are routinely performed under supra-physiologic (21% O2 , ambient air) conditions, which limits clinical translatability. We hereby identify molecular pathways enriched in AML cells that survive acute challenges with classic or targeted therapeutic agents. Experiments took into account variations in O2 tension encountered by leukaemic cells in clinical settings. Integrated RNA and protein profiles revealed that lipid biosynthesis, and particularly the cholesterol biogenesis branch, is a particularly therapy-induced vulnerability in AML cells under low O2 states. We also demonstrate that the impact of the cytotoxic agent cytarabine is selectively enhanced by a high-potency statin. The cholesterol biosynthesis programme is amenable to additional translational opportunities within the expanding AML therapeutic landscape. Our findings support the further investigation of higher-potency statin (eg rosuvastatin)-based combination therapies to enhance targeting residual AML cells that reside in low O2 environments.

Scavenger receptor class B, type I-mediated uptake of A1AT by pulmonary endothelial cells.

In addition to exerting a potent anti-elastase function, α-1 antitrypsin (A1AT) maintains the structural integrity of the lung by inhibiting endothelial inflammation and apoptosis. A main serpin secreted in circulation by hepatocytes, A1AT requires uptake by the endothelium to achieve vasculoprotective effects. This active uptake mechanism, which is inhibited by cigarette smoking (CS), involves primarily clathrin- but also caveola-mediated endocytosis and may require active binding to a receptor. Because circulating A1AT binds to high-density lipoprotein (HDL), we hypothesized that scavenging receptors are candidates for endothelial uptake of the serpin. Although the low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) internalizes only elastase-bound A1AT, the scavenger receptor B type I (SR-BI), which binds and internalizes HDL and is modulated by CS, may be involved in A1AT uptake. Transmission electron microscopy imaging of colloidal gold-labeled A1AT confirmed A1AT endocytosis in both clathrin-coated vesicles and caveolae in endothelial cells. SR-BI immunoprecipitation identified binding to A1AT at the plasma membrane. Pretreatment of human lung microvascular endothelial cells with SR-B ligands (HDL or LDL), knockdown of SCARB1 expression, or neutralizing SR-BI antibodies significantly reduced A1AT uptake by 30-50%. Scarb1 null mice exhibited decreased A1AT lung content following systemic A1AT administration and reduced lung anti-inflammatory effects of A1AT supplementation during short-term CS exposure. In turn, A1AT supplementation increased lung SR-BI expression and modulated circulating lipoprotein levels in wild-type animals. These studies indicate that SR-BI is an important mediator of A1AT endocytosis in pulmonary endothelium and suggest a cross talk between A1AT and lipoprotein regulation of vascular functions.

Effects of lipid interactions on model vesicle engulfment by alveolar macrophages.

The engulfment function of macrophages relies on complex molecular interactions involving both lipids and proteins. In particular, the clearance of apoptotic bodies (efferocytosis) is enabled by externalization on the cell target of phosphatidylserine lipids, which activate receptors on macrophages, suggesting that (local) specific lipid-protein interactions are required at least for the initiation of efferocytosis. However, in addition to apoptotic cells, macrophages can engulf foreign bodies that vary substantially in size from a few nanometers to microns, suggesting that nonspecific interactions over a wide range of length scales could be relevant. Here, we use model lipid membranes (made of phosphatidylcholine, phosphatidylserine, and ceramide) and rat alveolar macrophages to show how lipid bilayer properties probed by small-angle x-ray scattering and solid-state (2)H NMR correlate with engulfment rates measured by flow cytometry. We find that engulfment of protein-free model lipid vesicles is promoted by the presence of phosphatidylserine lipids but inhibited by ceramide, in accord with a previous study of apoptotic cells. We conclude that the roles of phosphatidylserine and ceramide in phagocytosis is based, at least in part, on lipid-mediated modification of membrane physical properties, including interactions at large length scales as well as local lipid ordering and possible domain formation.

Smoking exposure induces human lung endothelial cell adaptation to apoptotic stress.

Prolonged exposure to cigarette smoking is the main risk factor for emphysema, a component of chronic obstructive pulmonary diseases (COPDs) characterized by destruction of alveolar walls. Moreover, smoking is associated with pulmonary artery remodeling and pulmonary hypertension, even in the absence of COPD, through as yet unexplained mechanisms. In murine models, elevations of intra- and paracellular ceramides in response to smoking have been implicated in the induction of lung endothelial cell apoptosis, but the role of ceramides in human cell counterparts is yet unknown. We modeled paracrine increases (outside-in) of palmitoyl ceramide (Cer16) in primary human lung microvascular cells. In naive cells, isolated from nonsmokers, Cer16 significantly reduced cellular proliferation and induced caspase-independent apoptosis via mitochondrial membrane depolarization, apoptosis-inducing factor translocation, and poly(ADP-ribose) polymerase cleavage. In these cells, caspase-3 was inhibited by ceramide-induced Akt phosphorylation, and by the induction of autophagic microtubule-associated protein-1 light-chain 3 lipidation. In contrast, cells isolated from smokers exhibited increased baseline proliferative features associated with lack of p16(INK4a) expression and Akt hyperphosphorylation. These cells were resistant to Cer16-induced apoptosis, despite presence of both endoplasmic reticulum stress response and mitochondrial membrane depolarization. In cells from smokers, the prominent up-regulation of Akt pathways inhibited ceramide-triggered apoptosis, and was associated with elevated sphingosine and high-mobility group box 1, skewing the cell's response toward autophagy and survival. In conclusion, the cell responses to ceramide are modulated by an intricate cross-talk between Akt signaling and sphingolipid metabolites, and profoundly modified by previous cigarette smoke exposure, which selects for an apoptosis-resistant phenotype.

HIV envelope protein gp120-induced apoptosis in lung microvascular endothelial cells by concerted upregulation of EMAP II and its receptor, CXCR3.

Chronic lung diseases, such as pulmonary emphysema, are increasingly recognized complications of infection with the human immunodeficiency virus (HIV). Emphysema in HIV may occur independent of cigarette smoking, via mechanisms that are poorly understood but may involve lung endothelial cell apoptosis induced by the HIV envelope protein gp120. Recently, we have demonstrated that lung endothelial apoptosis is an important contributor to the development of experimental emphysema, via upregulation of the proinflammatory cytokine endothelial monocyte-activating polypeptide II (EMAP II) in the lung. Here we investigated the role of EMAP II and its receptor, CXCR3, in gp120-induced lung endothelial cell apoptosis. We could demonstrate that gp120 induces a rapid and robust increase in cell surface expression of EMAP II and its receptor CXCR3. This surface expression occurred via a mechanism involving gp120 signaling through its CXCR4 receptor and p38 MAPK activation. Both EMAP II and CXCR3 were essentially required for gp120-induced apoptosis and exposures to low gp120 concentrations enhanced the susceptibility of endothelial cells to undergo apoptosis when exposed to soluble cigarette smoke extract. These data indicate a novel mechanism by which HIV infection causes endothelial cell loss involved in lung emphysema formation, independent but potentially synergistic with smoking, and suggest therapeutic targets for emphysema prevention and/or treatment.

The involvement of sphingolipids in chronic obstructive pulmonary diseases.

Chronic obstructive pulmonary disease (COPD) includes a spectrum of conditions that have in common varying degrees of airflow obstruction, such as chronic bronchitis and emphysema. There is an increasing evidence of involvement of sphingolipids as key molecular mediators or biomarkers of disease in emphysema, chronic bronchitis, and more recently in asthma, another disease characterized by (reversible) airflow obstruction. Given the recognized central role of oxidative stress and inflammatory stimuli along with involvement of immune responses, apoptosis, and tissue remodeling in the development of chronic obstructive lung diseases, it is not surprising that sphingolipids have been shown to play important role in their pathobiology. In particular the pro-apoptotic effects of ceramide were suspected as events in the lung destruction that occurs as a result of apoptotic loss of structural cells comprising the alveolar walls, such as microvascular endothelial cells and alveolar epithelial cells. In addition, the role of ceramide was investigated in models of larger airway epithelial cell stress responses to cigarette smoke, in the context of ensuing airway remodeling and inflammation. This chapter discusses current evidence of sphingolipid perturbations in experimental models of COPD and relevant links to human disease based on translational and epidemiological data.

Cigarette smoke-induced CXCR3 receptor up-regulation mediates endothelial apoptosis.

Endothelial monocyte-activating polypeptide II (EMAP II) and interferon-inducible protein (IP)-10 are proinflammatory mediators, which in addition to their chemokine activities, selectively induce apoptosis in endothelial cells and are up-regulated in the lungs of cigarette smoke-exposed humans. Previously, we showed that EMAP II is an essential mediator of cigarette smoke-induced lung emphysema in mice linking endothelial cell apoptosis with inflammation. Here we addressed the role of the CXCR3 receptor in EMAP II-induced and IP-10-induced apoptosis in endothelial cells and its regulation by cigarette smoke. We found that both neutralizing antibodies and small inhibitory RNA to CXCR3 abrogated EMAP II-induced and IP-10-induced endothelial caspase-3 activation and DNA fragmentation. CXCR3 receptor surface expression in human lung microvascular endothelial cells and in lung tissue endothelium was up-regulated by exposure to cigarette smoke. In tissue culture conditions, EMAP II-induced and IP-10-induced apoptosis was enhanced by preincubation with cigarette smoke extract. Interestingly, serum starvation also induced CXCR3 up-regulation and enhanced EMAP II-induced endothelial apoptosis. Signal transduction via p38 mitogen-activated protein kinase activation was essential for CXCR3-induced cell death, but not for CXCR3 receptor up-regulation by cigarette smoke. In turn, protein nitration was required for CXCR3 receptor up-regulation by cigarette smoke and consequently for subsequent CXCR3-induced cell death. In conclusion, the concerted up-regulation of proinflammatory EMAP II, IP-10, and CXCR3 by cigarette smoke could sustain a cascade of cell death that may promote the alveolar tissue loss noted in human emphysema.

Role of Sphingolipids in Multiple Myeloma Progression, Drug Resistance, and Their Potential as Therapeutic Targets.

Multiple myeloma (MM) is an incapacitating hematological malignancy characterized by accumulation of cancerous plasma cells in the bone marrow (BM) and production of an abnormal monoclonal protein (M-protein). The BM microenvironment has a key role in myeloma development by facilitating the growth of the aberrant plasma cells, which eventually interfere with the homeostasis of the bone cells, exacerbating osteolysis and inhibiting osteoblast differentiation. Recent recognition that metabolic reprograming has a major role in tumor growth and adaptation to specific changes in the microenvironmental niche have led to consideration of the role of sphingolipids and the enzymes that control their biosynthesis and degradation as critical mediators of cancer since these bioactive lipids have been directly linked to the control of cell growth, proliferation, and apoptosis, among other cellular functions. In this review, we present the recent progress of the research investigating the biological implications of sphingolipid metabolism alterations in the regulation of myeloma development and its progression from the pre-malignant stage and discuss the roles of sphingolipids in in MM migration and adhesion, survival and proliferation, as well as angiogenesis and invasion. We introduce the current knowledge regarding the role of sphingolipids as mediators of the immune response and drug-resistance in MM and tackle the new developments suggesting the manipulation of the sphingolipid network as a novel therapeutic direction for MM.

GFI1-Dependent Repression of SGPP1 Increases Multiple Myeloma Cell Survival.

Multiple myeloma (MM) remains incurable for most patients due to the emergence of drug resistant clones. Here we report a p53-independent mechanism responsible for Growth Factor Independence-1 (GFI1) support of MM cell survival by its modulation of sphingolipid metabolism to increase the sphingosine-1-phosphate (S1P) level regardless of the p53 status. We found that expression of enzymes that control S1P biosynthesis, SphK1, dephosphorylation, and SGPP1 were differentially correlated with GFI1 levels in MM cells. We detected GFI1 occupancy on the SGGP1 gene in MM cells in a predicted enhancer region at the 5' end of intron 1, which correlated with decreased SGGP1 expression and increased S1P levels in GFI1 overexpressing cells, regardless of their p53 status. The high S1P:Ceramide intracellular ratio in MM cells protected c-Myc protein stability in a PP2A-dependent manner. The decreased MM viability by SphK1 inhibition was dependent on the induction of autophagy in both p53WT and p53mut MM. An autophagic blockade prevented GFI1 support for viability only in p53mut MM, demonstrating that GFI1 increases MM cell survival via both p53WT inhibition and upregulation of S1P independently. Therefore, GFI1 may be a key therapeutic target for all types of MM that may significantly benefit patients that are highly resistant to current therapies.

Sphingolipid-mediated inhibition of apoptotic cell clearance by alveolar macrophages.

A decreased clearance of apoptotic cells (efferocytosis) by alveolar macrophages (AM) may contribute to inflammation in emphysema. The up-regulation of ceramides in response to cigarette smoking (CS) has been linked to AM accumulation and increased detection of apoptotic alveolar epithelial and endothelial cells in lung parenchyma. We hypothesized that ceramides inhibit the AM phagocytosis of apoptotic cells. Release of endogenous ceramides via sphingomyelinase or exogenous ceramide treatments dose-dependently impaired apoptotic Jurkat cell phagocytosis by primary rat or human AM, irrespective of the molecular species of ceramide. Similarly, in vivo augmentation of lung ceramides via intratracheal instillation in rats significantly decreased the engulfment of instilled target apoptotic thymocytes by resident AM. The mechanism of ceramide-induced efferocytosis impairment was dependent on generation of sphingosine via ceramidase. Sphingosine treatment recapitulated the effects of ceramide, dose-dependently inhibiting apoptotic cell clearance. The effect of ceramide on efferocytosis was associated with decreased membrane ruffle formation and attenuated Rac1 plasma membrane recruitment. Constitutively active Rac1 overexpression rescued AM efferocytosis against the effects of ceramide. CS exposure significantly increased AM ceramides and recapitulated the effect of ceramides on Rac1 membrane recruitment in a sphingosine-dependent manner. Importantly, CS profoundly inhibited AM efferocytosis via ceramide-dependent sphingosine production. These results suggest that excessive lung ceramides may amplify lung injury in emphysema by causing both apoptosis of structural cells and inhibition of their clearance by AM.

Therapeutic targets in myeloma bone disease.

Multiple myeloma (MM) is the second most common haematological malignancy and is characterized by a clonal proliferation of neoplastic plasma cells within the bone marrow. MM is the most frequent cancer involving the skeleton, causing osteolytic lesions, bone pain and pathological fractures that dramatically decrease MM patients' quality of life and survival. MM bone disease (MBD) results from uncoupling of bone remodelling in which excessive bone resorption is not compensated by new bone formation, due to a persistent suppression of osteoblast activity. Current management of MBD includes antiresorptive agents, bisphosphonates and denosumab, that are only partially effective due to their inability to repair the existing lesions. Thus, research into agents that prevent bone destruction and more importantly repair existing lesions by inducing new bone formation is essential. This review discusses the mechanisms regulating the uncoupled bone remodelling in MM and summarizes current advances in the treatment of MBD. LINKED ARTICLES: This article is part of a themed issue on The molecular pharmacology of bone and cancer-related bone diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.9/issuetoc.

Mechanism of alpha-1 antitrypsin endocytosis by lung endothelium.

The integrity of lung alveoli is maintained by proper circulating levels of alpha-1 antitrypsin (A1AT). Next to cigarette smoking, A1AT deficiency is a major risk factor for lung emphysema development. We recently reported that in addition to neutralizing neutrophil elastases in the extracellular compartment, A1AT is internalized by lung endothelial cells and inhibits apoptosis. We hypothesized that the intracellular uptake of A1AT by endothelial cells may be required for its protective function; therefore, we studied the mechanisms of A1AT internalization by primary rat lung microvascular endothelial cells and the effect of cigarette smoke on this process both in vitro and in vivo (in mice). Purified A1AT was taken up intracellularly by endothelial cells in a time-dependent, dose-dependent, and conformer-specific manner and was detected in the cytoplasm of endothelial cells of nondiseased human lung sections. Despite a critical role for caveoli in endothelial cell endocytosis in general, specific inhibition of clathrin-mediated, but not caveoli-mediated, endocytosis profoundly decreased A1AT internalization and reversed the A1AT's antiapoptotic action. Further more, A1AT associated with clathrin heavy chains, but not with caveolin-1 in the plasma membrane fraction of endothelial cells. Interestingly, cigarette smoke exposure significantly inhibited A1AT uptake both in endothelial cells and in the mouse lung and altered the intracellular distribution of clathrin heavy chains. Our results suggest that clathrin-mediated endocytosis regulates A1AT intracellular function in the lung endothelium and may be an important determinant of the serpin's protection against developing cigarette smoke-induced emphysema.

Osteocyte Vegf-a contributes to myeloma-associated angiogenesis and is regulated by Fgf23.

Multiple Myeloma (MM) induces bone destruction, decreases bone formation, and increases marrow angiogenesis in patients. We reported that osteocytes (Ocys) directly interact with MM cells to increase tumor growth and expression of Ocy-derived factors that promote bone resorption and suppress bone formation. However, the contribution of Ocys to enhanced marrow vascularization in MM is unclear. Since the MM microenvironment is hypoxic, we assessed if hypoxia and/or interactions with MM cells increases pro-angiogenic signaling in Ocys. Hypoxia and/or co-culture with MM cells significantly increased Vegf-a expression in MLOA5-Ocys, and conditioned media (CM) from MLOA5s or MM-MLOA5 co-cultured in hypoxia, significantly increased endothelial tube length compared to normoxic CM. Further, Vegf-a knockdown in MLOA5s or primary Ocys co-cultured with MM cells or neutralizing Vegf-a in MM-Ocy co-culture CM completely blocked the increased endothelial activity. Importantly, Vegf-a-expressing Ocy numbers were significantly increased in MM-injected mouse bones, positively correlating with tumor vessel area. Finally, we demonstrate that direct contact with MM cells increases Ocy Fgf23, which enhanced Vegf-a expression in Ocys. Fgf23 deletion in Ocys blocked these changes. These results suggest hypoxia and MM cells induce a pro-angiogenic phenotype in Ocys via Fgf23 and Vegf-a signaling, which can promote MM-induced marrow vascularization.

Publisher Correction: Regulation of cellular sterol homeostasis by the oxygen responsive noncoding RNA lincNORS.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Regulation of cellular sterol homeostasis by the oxygen responsive noncoding RNA lincNORS.

We hereby provide the initial portrait of lincNORS, a spliced lincRNA generated by the MIR193BHG locus, entirely distinct from the previously described miR-193b-365a tandem. While inducible by low O2 in a variety of cells and associated with hypoxia in vivo, our studies show that lincNORS is subject to multiple regulatory inputs, including estrogen signals. Biochemically, this lincRNA fine-tunes cellular sterol/steroid biosynthesis by repressing the expression of multiple pathway components. Mechanistically, the function of lincNORS requires the presence of RALY, an RNA-binding protein recently found to be implicated in cholesterol homeostasis. We also noticed the proximity between this locus and naturally occurring genetic variations highly significant for sterol/steroid-related phenotypes, in particular the age of sexual maturation. An integrative analysis of these variants provided a more formal link between these phenotypes and lincNORS, further strengthening the case for its biological relevance.

CFTR regulation of intracellular pH and ceramides is required for lung endothelial cell apoptosis.

The functional significance of the expression of cystic fibrosis transmembrane regulator (CFTR) on endothelial cells has not yet been elucidated. Since CFTR has been implicated in the regulation of intracellular sphingolipid levels, which are important regulators of endothelial cell apoptosis in response to various insults, we investigated the role of CFTR in the apoptotic responses of lung endothelial cells. CFTR was detected as a functional chloride channel in primary lung endothelial cells isolated from both pulmonary arteries (human or mouse) and bronchial arteries (sheep). Both specific CFTR inhibition with 2-(phenylamino) benzoic acid diphenylamine-2-carboxylic acid, 5-[(4-carboxyphenyl)methylene]-2-thioxo-3-[(3-trifluoromethyl)phenyl-4-thiazolidinone (CFTR(inh)-172), or 5-nitro-2-(3-phenylpropylamino)benzoic acid and CFTR knockdown significantly attenuated endothelial cell apoptosis induced by staurosporine or H(2)O(2). CFTR(inh)-172 treatment prevented the increases in the ceramide:sphingosine-1 phosphate ratio induced by H(2)O(2) in lung endothelial cells. Replenishing endogenous ceramides via sphingomyelinase supplementation restored the susceptibility of CFTR-inhibited lung endothelial cells to H(2)O(2)-induced apoptosis. Similarly, the anti-apoptotic phenotype of CFTR-inhibited cells was reversed by lowering the intracellular pH, and was reproduced by alkalinization before H(2)O(2) challenge. TUNEL staining and active caspase-3 immunohistochemistry indicated that cellular apoptosis was decreased in lung explants from patients with cystic fibrosis compared with those with smoking-induced chronic obstructive lung disease, especially in the alveolar tissue and vascular endothelium. In conclusion, CFTR function is required for stress-induced apoptosis in lung endothelial cells by maintaining adequate intracellular acidification and ceramide activation. These results may have implications in the pathogenesis of cystic fibrosis, where aberrant endothelial cell death may dysregulate lung vascular homeostasis, contributing to abnormal angiogenesis and chronic inflammation.

Spinal ceramide modulates the development of morphine antinociceptive tolerance via peroxynitrite-mediated nitroxidative stress and neuroimmune activation.

The effective treatment of pain is typically limited by a decrease in the pain-relieving action of morphine that follows its chronic administration (tolerance). Therefore, restoring opioid efficacy is of great clinical importance. In a murine model of opioid antinociceptive tolerance, repeated administration of morphine significantly stimulated the enzymatic activities of spinal cord serine palmitoyltransferase, ceramide synthase, and acid sphingomyelinase (enzymes involved in the de novo and sphingomyelinase pathways of ceramide biosynthesis, respectively) and led to peroxynitrite-derive nitroxidative stress and neuroimmune activation [activation of spinal glial cells and increase formation of tumor necrosis factor-alpha, interleukin (IL)-1beta, and IL-6]. Inhibition of ceramide biosynthesis with various pharmacological inhibitors significantly attenuated the increase in spinal ceramide production, nitroxidative stress, and neuroimmune activation. These events culminated in a significant inhibition of the development of morphine antinociceptive tolerance at doses devoid of behavioral side effects. Our findings implicate ceramide as a key upstream signaling molecule in the development of morphine antinociceptive tolerance and provide the rationale for development of inhibitors of ceramide biosynthesis as adjuncts to opiates for the management of chronic pain.