False colored section of mouse colon prior to treatment with Gamitrinibs, a class of small molecules designed by Kang and colleagues (page 454) to selectively target the heat shock protein-90 (Hsp90) network compartmentalized in tumor mitochondria. Normal cells and tissues, and global Hsp90 outside of the mitochondria, were unaffected.
An inauspicious start to 2009, unfortunately. This issue may seem a little thinner than others we’ve recently published, as 4 articles that were previously accepted and scheduled for publication in this issue will not appear. We continue to screen all figures from accepted manuscripts, and we continue to find irregularities. In several cases, the alterations in the figures led to the discovery of some fundamental problems with the data. Many of the papers suffered from the same problems, and this led us to consider whether it was time to revisit some experimental basics.
Bardet-Biedl syndrome (BBS) is a multisystemic disorder typified by developmental and progressive degenerative defects. A combination of genetic, in vitro, and in vivo studies have highlighted ciliary dysfunction as a primary cause of BBS pathology, which has in turn contributed to the improved understanding of the functions of the primary cilium in humans and other vertebrates. Here we discuss the evidence linking the clinical BBS phenotype to ciliary defects, highlight how the genetic and cellular characteristics of BBS overlap with and inform other ciliary disorders, and explore the possible mechanistic underpinnings of ciliary dysfunction.
In vertebrates, the detection of viral nucleic acids is the first step toward innate and subsequent adaptive antiviral immune responses. A sophisticated, protein receptor–based sensor system has evolved to recognize viral nucleic acids and to trigger a variety of antiviral defense mechanisms. The more we learn about this elaborate sensor system, the more it becomes evident how difficult it is to introduce exogenous nucleic acids such as siRNA into cells without triggering antiviral immunoreceptors. In this issue of the JCI, Judge and colleagues provide evidence that siRNA can be designed and delivered in a way that allows specific and successful silencing of target genes in tumor cells in vivo, leading to tumor cell death and prolonged survival of tumor-bearing mice in the absence of immune activation (see the related article beginning on page 661). This study represents a major technological advance, setting new standards for well-controlled siRNA applications in vivo, and has the potential to guide clinical development toward siRNA therapeutics with well-defined and selective gene-silencing activities.
Mutations in the genes PTEN-induced putative kinase 1 (PINK1), PARKIN, and DJ-1 cause autosomal recessive forms of Parkinson disease (PD), and the Pink1/Parkin pathway regulates mitochondrial integrity and function. An important question is whether the proteins encoded by these genes function to regulate activities of other cellular compartments. A study in mice, reported by Xiong et al. in this issue of the JCI, demonstrates that Pink1, Parkin, and DJ-1 can form a complex in the cytoplasm, with Pink1 and DJ-1 promoting the E3 ubiquitin ligase activity of Parkin to degrade substrates via the proteasome (see the related article beginning on page 650). This protein complex in the cytosol may or may not be related to the role of these proteins in regulating mitochondrial function or oxidative stress in vivo.
The targeting of tumors is made possible through establishing protein signatures specific for each cancer type. The recent recognition of the higher expression levels of HSP90 and its accumulation in tumor cell mitochondria has made the HSP90 network a feasible target for neutralization. HSP90 antagonizes the mitochondrial permeability transition, blocking cytochrome c release and apoptosis. In this issue of the JCI, Kang et al. report the synthesis of Gamitrinibs, which target mitochondrially localized HSP90, specifically killing human cancer cell lines, and provide a fresh approach for cancer treatment (see the related article beginning on page 454).
NOS is a key enzyme in the production of NO, a molecule that directly regulates vasorelaxation and blood supply. Diverse forms of muscle disease have been clinically associated with unusual fatigue after exercise. The localization of neuronal NOS (nNOS) at the plasma membrane of muscle has recently been shown to prevent muscle fatigue after exercise. In this issue of the JCI, Lai et al. show that dystrophin — the structural protein missing in individuals with Duchenne muscular dystrophy — anchors nNOS to the sarcolemma through a direct interaction with dystrophin spectrin-like repeats 16 and 17 (see the related article beginning on page 624). Furthermore, in another recently reported study of mouse models of muscular dystrophy, phosphodiesterase 5A inhibitors were used to treat the downstream ischemia that is associated with nNOS mislocalization. Collectively, these findings significantly advance our understanding of exercise-induced muscle fatigue and its role in muscle disease.
Adaptive T cell responses are critical for controlling infections with viruses such as HIV, HBV, and HCV. However, these responses must be carefully regulated because overly vigorous T cell activation can lead to excessive host tissue damage. T cell expression of the inhibitory receptor programmed death–1 (PD-1) and inhibition of effector T cells (Teffs) by CD4+Foxp3+ Tregs are among the many described mechanisms for achieving a balanced immune response. Although the signals that contribute to Teff function are well understood, less is known about the signals controlling Tregs. In this issue of the JCI, Franceschini et al. extend our understanding of how Tregs are modulated during chronic HCV infection by demonstrating that Treg proliferation is inhibited by PD-1 and that this inhibition is mediated by a potentially novel mechanism involving the prevention of IL-2–driven STAT-5 phosphorylation (see the related article beginning on page 551).
Although therapeutically targeting a single signaling pathway that drives tumor development and/or progression has been effective for a number of cancers, in many cases this approach has not been successful. Targeting networks of signaling pathways, instead of isolated pathways, may overcome this problem, which is probably due to the extreme heterogeneity of human tumors. However, the possibility that such networks may be spatially arranged in specialized subcellular compartments is not often considered in pathway-oriented drug discovery and may influence the design of new agents. Hsp90 is a chaperone protein that controls the folding of proteins in multiple signaling networks that drive tumor development and progression. Here, we report the synthesis and properties of Gamitrinibs, a class of small molecules designed to selectively target Hsp90 in human tumor mitochondria. Gamitrinibs were shown to accumulate in the mitochondria of human tumor cell lines and to inhibit Hsp90 activity by acting as ATPase antagonists. Unlike Hsp90 antagonists not targeted to mitochondria, Gamitrinibs exhibited a “mitochondriotoxic” mechanism of action, causing rapid tumor cell death and inhibiting the growth of xenografted human tumor cell lines in mice. Importantly, Gamitrinibs were not toxic to normal cells or tissues and did not affect Hsp90 homeostasis in cellular compartments other than mitochondria. Therefore, combinatorial drug design, whereby inhibitors of signaling networks are targeted to specific subcellular compartments, may generate effective anticancer drugs with novel mechanisms of action.
Hepatocellular carcinoma (HCC) is a highly aggressive vascular cancer characterized by diverse etiology, activation of multiple signal transduction pathways, and various gene mutations. Here, we have determined a specific role for astrocyte elevated gene-1 (AEG1) in HCC pathogenesis. Expression of AEG1 was extremely low in human hepatocytes, but its levels were significantly increased in human HCC. Stable overexpression of AEG1 converted nontumorigenic human HCC cells into highly aggressive vascular tumors, and inhibition of AEG1 abrogated tumorigenesis by aggressive HCC cells in a xenograft model of nude mice. In human HCC, AEG1 overexpression was associated with elevated copy numbers. Microarray analysis revealed that AEG1 modulated the expression of genes associated with invasion, metastasis, chemoresistance, angiogenesis, and senescence. AEG1 also was found to activate Wnt/β-catenin signaling via ERK42/44 activation and upregulated lymphoid-enhancing factor 1/T cell factor 1 (LEF1/TCF1), the ultimate executor of the Wnt pathway, important for HCC progression. Inhibition studies further demonstrated that activation of Wnt signaling played a key role in mediating AEG1 function. AEG1 also activated the NF-κB pathway, which may play a role in the chronic inflammatory changes preceding HCC development. These data indicate that AEG1 plays a central role in regulating diverse aspects of HCC pathogenesis. Targeted inhibition of AEG1 might lead to the shutdown of key elemental characteristics of HCC and could lead to an effective therapeutic strategy for HCC.
The HGF gene is transcriptionally silenced in normal differentiated breast epithelial cells, but its repression fails to occur in mammary carcinoma tissues and cell lines. The molecular mechanisms underpinning aberrant HGF expression in breast cancer cells are unknown. Here we report the discovery of a DNA element located 750 bp upstream from the transcription start site in the human HGF promoter that acts as a transcriptional repressor and is a target of deletion mutagenesis in human breast cancer cells and tissues. This HGF promoter element consists of a mononucleotide repeat of 30 deoxyadenosines (30As), which we have termed “deoxyadenosine tract element” (DATE). Functional studies revealed that truncation mutations within DATE have profound local and global effects on the HGF promoter region by modulating chromatin structure and DNA-protein interactions, leading to constitutive activation of the HGF promoter in human breast carcinoma cell lines. We found that 51% of African Americans and 15% of individuals of mixed European descent with breast cancer harbor a truncated DATE variant (25As or fewer) in their breast tumors and that the truncated allele is associated with cancer incidence and aberrant HGF expression. Notably, breast cancer patients with the truncated DATE variant are substantially younger than those with a wild-type genotype. We also suggest that DATE may be used as a potential genetic marker to identify individuals with a higher risk of developing breast cancer.
The mechanisms governing hematopoietic progenitor cell mobilization are not fully understood. We report higher membrane type 1–MMP (MT1-MMP) and lower expression of the MT1-MMP inhibitor, reversion-inducing cysteine-rich protein with Kazal motifs (RECK), on isolated circulating human CD34+ progenitor cells compared with immature BM cells. The expression of MT1-MMP correlated with clinical mobilization of CD34+ cells in healthy donors and patients with lymphoid malignancies. Treatment with G-CSF further increased MT1-MMP and decreased RECK expression in human and murine hematopoietic cells in a PI3K/Akt-dependent manner, resulting in elevated MT1-MMP activity. Blocking MT1-MMP function by Abs or siRNAs impaired chemotaxis and homing of G-CSF–mobilized human CD34+ progenitors. The mobilization of immature and maturing human progenitors in chimeric NOD/SCID mice by G-CSF was inhibited by anti–MT1-MMP treatment, while RECK neutralization promoted motility and egress of BM CD34+ cells. BM c-kit+ cells from MT1-MMP–deficient mice also exhibited inferior chemotaxis, reduced homing and engraftment capacities, and impaired G-CSF–induced mobilization in murine chimeras. Membranal CD44 cleavage by MT1-MMP was enhanced following G-CSF treatment, reducing CD34+ cell adhesion. Accordingly, CD44-deficient mice had a higher frequency of circulating progenitors. Our results reveal that the motility, adhesion, homing, and mobilization of human hematopoietic progenitor cells are regulated in a cell-autonomous manner by dynamic and opposite changes in MT1-MMP and RECK expression.
Thrombocytopenia and thrombosis following treatment with the integrin αIIbβ3 antagonist eptifibatide are rare complications caused by patient antibodies specific for ligand-occupied αIIbβ3. Whether such antibodies induce platelet clearance by simple opsonization, by inducing mild platelet activation, or both is poorly understood. To gain insight into the mechanism by which eptifibatide-dependent antibodies initiate platelet clearance, we incubated normal human platelets with patient serum containing an αIIbβ3-specific, eptifibatide-dependent antibody. We observed that in the presence of eptifibatide, patient IgG induced platelet secretion and aggregation as well as tyrosine phosphorylation of the integrin β3 cytoplasmic domain, the platelet FcγRIIa Fc receptor, the protein-tyrosine kinase Syk, and phospholipase Cγ2. Each activation event was inhibited by preincubation of the platelets with Fab fragments of the FcγRIIa-specific mAb IV.3 or with the Src family kinase inhibitor PP2. Patient serum plus eptifibatide did not, however, activate platelets from a patient with a variant form of Glanzmann thrombasthenia that expressed normal levels of FcγRIIa and the αIIbβ3 complex but lacked most of the β3 cytoplasmic domain. Taken together, these data suggest a novel mechanism whereby eptifibatide-dependent antibodies engage the integrin β3 subunit such that FcγRIIa and its downstream signaling components become activated, resulting in thrombocytopenia and a predisposition to thrombosis.
Pulmonary hypertension (PH) is a progressive, lethal lung disease characterized by pulmonary artery SMC (PA-SMC) hyperplasia leading to right-sided heart failure. Molecular events originating in pulmonary ECs (P-ECs) may contribute to the PA-SMC hyperplasia in PH. Thus, we exposed cultured human PA-SMC to medium conditioned by P-EC from patients with idiopathic PH (IPH) or controls and found that IPH P-EC–conditioned medium increased PA-SMC proliferation more than control P-EC medium. Levels of FGF2 were increased in the medium of IPH P-ECs over controls, while there was no detectable difference in TGF-β1, PDGF-BB, or EGF levels. No difference in FGF2-induced proliferation or FGF receptor type 1 (FGFR1) mRNA levels was detected between IPH and control PA-SMCs. Knockdown of FGF2 in P-EC using siRNA reduced the PA-SMC growth-stimulating effects of IPH P-EC medium by 60% and control P-EC medium by 10%. In situ hybridization showed FGF2 overproduction predominantly in the remodeled vascular endothelium of lungs from patients with IPH. Repeated intravenous FGF2-siRNA administration abolished lung FGF2 production, both preventing and nearly reversing a rat model of PH. Similarly, pharmacological FGFR1 inhibition with SU5402 reversed established PH in the same model. Thus, endothelial FGF2 is overproduced in IPH and contributes to SMC hyperplasia in IPH, identifying FGF2 as a promising target for new treatments against PH.
The renin-angiotensin system plays a role in the etiology of hypertension and the pathophysiology of cardiac and renal diseases in humans. Ang II is the central product of this system and is involved in regulating immune responses, inflammation, cell growth, and proliferation by acting through Ang II type 1 receptors (AT1 and AT2). Here, we show that targeted disruption of the Agtr1a gene that encodes AT1A results in marked prolongation of life span in mice. Agtr1a–/– mice developed less cardiac and vascular injury, and multiple organs from these mice displayed less oxidative damage than wild-type mice. The longevity phenotype was associated with an increased number of mitochondria and upregulation of the prosurvival genes nicotinamide phosphoribosyltransferase (Nampt) and sirtuin 3 (Sirt3) in the kidney. In cultured tubular epithelial cells, Ang II downregulated Sirt3 mRNA, and this effect was inhibited by an AT1 antagonist. These results demonstrate that disruption of AT1 promotes longevity in mice, possibly through the attenuation of oxidative stress and overexpression of prosurvival genes, and suggests that the Ang II/AT1 pathway may be targeted to influence life span in mammals.
Resistin is an adipokine that contributes to insulin resistance in mice. In humans, however, studies investigating the link between resistin and metabolic disease are conflicting. Further complicating the matter, human resistin is produced mainly by macrophages rather than adipocytes. To address this important issue, we generated mice that lack adipocyte-derived mouse resistin but produce human resistin in a pattern similar to that found in humans, i.e., in macrophages (humanized resistin mice). When placed on a high-fat diet, the humanized resistin mice rapidly developed accelerated white adipose tissue (WAT) inflammation, leading to increased lipolysis and increased serum free fatty acids. Over time, these mice accumulated lipids, including diacylglycerols, in muscle. We found that this resulted in increased Pkcq pathway activity, leading to increased serine phosphorylation of Irs-1 and insulin resistance. Thus, although the site of resistin production differs between species, human resistin exacerbates WAT inflammation and contributes to insulin resistance.
The epithelial anion channel CFTR interacts with multiple PDZ domain–containing proteins. Heterologous expression studies have demonstrated that the Na+/H+ exchanger regulatory factors, NHERF1, NHERF2, and PDZK1 (NHERF3), modulate CFTR membrane retention, conductivity, and interactions with other transporters. To study their biological roles in vivo, we investigated CFTR-dependent duodenal HCO3– secretion in mouse models of Nherf1, Nherf2, and Pdzk1 loss of function. We found that Nherf1 ablation strongly reduced basal as well as forskolin-stimulated (FSK-stimulated) HCO3– secretory rates and blocked β2-adrenergic receptor (β2-AR) stimulation. Conversely, Nherf2–/– mice displayed augmented FSK-stimulated HCO3– secretion. Furthermore, although lysophosphatidic acid (LPA) inhibited FSK-stimulated HCO3– secretion in WT mice, this effect was lost in Nherf2–/– mice. Pdzk1 ablation reduced basal, but not FSK-stimulated, HCO3– secretion. In addition, laser microdissection and quantitative PCR revealed that the β2-AR and the type 2 LPA receptor were expressed together with CFTR in duodenal crypts and that colocalization of the β2-AR and CFTR was reduced in the Nherf1–/– mice. These data suggest that the NHERF proteins differentially modulate duodenal HCO3– secretion: while NHERF1 is an obligatory linker for β2-AR stimulation of CFTR, NHERF2 confers inhibitory signals by coupling the LPA receptor to CFTR.
CD4+CD25+Foxp3+ Tregs suppress autoimmune responses. In addition, they limit T cell responses during chronic infection, thereby minimizing T cell–dependent immunopathology. We sought to investigate how Tregs are regulated in the livers of patients chronically infected with HCV, where they control the balance between an adequate protective immune response and suppression of immunopathology. We found that, despite accumulating and proliferating at sites of infection in the livers of patients chronically infected with HCV, Tregs were relatively less expanded than CD4+CD25+Foxp3– effector T cells. The relative lower expansion of intrahepatic Tregs coincided with their upregulation of programmed death–1 (PD-1). PD-1 expression inversely correlated with both Treg proliferation and clinical markers of immune suppression in vivo. Consistent with the possibility that PD-1 controls Tregs, blockade of the interaction between PD-1 and programmed death–1 ligand 1 (PD-L1) enhanced the in vitro expansion and function of Tregs isolated from the livers of patients chronically infected with HCV. Blockade of the interaction between PD-L1 and B7.1 also improved the proliferation of these cells. Interestingly, both PD-1 and phosphorylated STAT-5 were overexpressed in intrahepatic Tregs in a parallel fashion in steady disease conditions, and in an alternate-fluctuating fashion during the course of severe hepatitis reactivation. Notably, PD-L1 blockade upregulated STAT-5 phosphorylation in Tregs ex vivo. These data suggest that PD-L1 negatively regulates Tregs at sites of chronic inflammation by controlling STAT-5 phosphorylation.
Th17 cells are involved in the pathogenesis of many autoimmune diseases, but it is not clear whether they play a pathogenic role in type 1 diabetes. Here we investigated whether mouse Th17 cells with specificity for an islet antigen can induce diabetes upon transfer into NOD/SCID recipient mice. Induction of diabetes in NOD/SCID mice via adoptive transfer of Th1 cells from BDC2.5 transgenic mice was prevented by treatment of the recipient mice with a neutralizing IFN-γ–specific antibody. This result suggested a major role of Th1 cells in the induction of disease in this model of type 1 diabetes. Nevertheless, transfer of highly purified Th17 cells from BDC2.5 transgenic mice caused diabetes in NOD/SCID recipients with similar rates of onset as in transfer of Th1 cells. However, treatment with neutralizing IL-17–specific antibodies did not prevent disease. Instead, the transferred Th17 cells, completely devoid of IFN-γ at the time of transfer, rapidly converted to secrete IFN-γ in the NOD/SCID recipients. Purified Th17 cells also upregulated Tbet and secreted IFN-γ upon exposure to IL-12 in vitro and in vivo in NOD/SCID recipients. These results indicate substantial plasticity of Th17 commitment toward a Th1-like profile.
High dietary fat intake leads to insulin resistance in skeletal muscle, and this represents a major risk factor for type 2 diabetes and cardiovascular disease. Mitochondrial dysfunction and oxidative stress have been implicated in the disease process, but the underlying mechanisms are still unknown. Here we show that in skeletal muscle of both rodents and humans, a diet high in fat increases the H2O2-emitting potential of mitochondria, shifts the cellular redox environment to a more oxidized state, and decreases the redox-buffering capacity in the absence of any change in mitochondrial respiratory function. Furthermore, we show that attenuating mitochondrial H2O2 emission, either by treating rats with a mitochondrial-targeted antioxidant or by genetically engineering the overexpression of catalase in mitochondria of muscle in mice, completely preserves insulin sensitivity despite a high-fat diet. These findings place the etiology of insulin resistance in the context of mitochondrial bioenergetics by demonstrating that mitochondrial H2O2 emission serves as both a gauge of energy balance and a regulator of cellular redox environment, linking intracellular metabolic balance to the control of insulin sensitivity.
Fatty liver is commonly associated with alcohol ingestion and abuse. While the molecular pathogenesis of these fatty changes is well understood, the biochemical and pharmacological mechanisms by which ethanol stimulates these molecular changes remain unknown. During ethanol metabolism, adenosine is generated by the enzyme ecto-5′-nucleotidase, and adenosine production and adenosine receptor activation are known to play critical roles in the development of hepatic fibrosis. We therefore investigated whether adenosine and its receptors play a role in the development of alcohol-induced fatty liver. WT mice fed ethanol on the Lieber-DeCarli diet developed hepatic steatosis, including increased hepatic triglyceride content, while mice lacking ecto-5′-nucleotidase or adenosine A1 or A2B receptors were protected from developing fatty liver. Similar protection was also seen in WT mice treated with either an adenosine A1 or A2B receptor antagonist. Steatotic livers demonstrated increased expression of genes involved in fatty acid synthesis, which was prevented by blockade of adenosine A1 receptors, and decreased expression of genes involved in fatty acid metabolism, which was prevented by blockade of adenosine A2B receptors. In vitro studies supported roles for adenosine A1 receptors in promoting fatty acid synthesis and for A2B receptors in decreasing fatty acid metabolism. These results indicate that adenosine generated by ethanol metabolism plays an important role in ethanol-induced hepatic steatosis via both A1 and A2B receptors and suggest that targeting adenosine receptors may be effective in the prevention of alcohol-induced fatty liver.
Anti-GM1 ganglioside autoantibodies are used as diagnostic markers for motor axonal peripheral neuropathies and are believed to be the primary mediators of such diseases. However, their ability to bind and exert pathogenic effects at neuronal membranes is highly inconsistent. Using human and mouse monoclonal anti-GM1 antibodies to probe the GM1-rich motor nerve terminal membrane in mice, we here show that the antigenic oligosaccharide of GM1 in the live plasma membrane is cryptic, hidden on surface domains that become buried for a proportion of anti-GM1 antibodies due to a masking effect of neighboring gangliosides. The cryptic GM1 binding domain was exposed by sialidase treatment that liberated sialic acid from masking gangliosides including GD1a or by disruption of the live membrane by freezing or fixation. This cryptic behavior was also recapitulated in solid-phase immunoassays. These data show that certain anti-GM1 antibodies exert potent complement activation-mediated neuropathogenic effects, including morphological damage at living terminal motor axons, leading to a block of synaptic transmission. This occurred only when GM1 was topologically available for antibody binding, but not when GM1 was cryptic. This revised understanding of the complexities in ganglioside membrane topology provides a mechanistic account for wide variations in the neuropathic potential of anti-GM1 antibodies.
In several disease states, abnormal growth of blood vessels is associated with local neuronal degeneration. This is particularly true in ocular diseases such as retinal angiomatous proliferation (RAP) and macular telangiectasia (MacTel), in which, despite the absence of large-scale leakage or hemorrhage, abnormal neovascularization (NV) is associated with local neuronal dysfunction. We describe here a retinal phenotype in mice with dysfunctional receptors for VLDL (Vldlr–/– mice) that closely resembles human retinal diseases in which abnormal intra- and subretinal NV is associated with photoreceptor cell death. Such cell death was evidenced by decreased cone and, to a lesser extent, rod opsin expression and abnormal electroretinograms. Cell death in the region of intraretinal vascular abnormalities was associated with an increased presence of markers associated with oxidative stress. Oral antioxidant supplementation protected against photoreceptor degeneration and preserved retinal function, despite the continued presence of abnormal intra- and subretinal vessels. What we believe to be novel, Müller cell–based, virally mediated delivery of neurotrophic compounds specifically to sites of NV was also neuroprotective. These observations demonstrate that neuronal loss secondary to NV can be prevented by the use of simple antioxidant dietary measures or cell-based delivery of neurotrophic factors, even when the underlying vascular phenotype is not altered.
Sarcolemma-associated neuronal NOS (nNOS) plays a critical role in normal muscle physiology. In Duchenne muscular dystrophy (DMD), the loss of sarcolemmal nNOS leads to functional ischemia and muscle damage; however, the mechanism of nNOS subcellular localization remains incompletely understood. According to the prevailing model, nNOS is recruited to the sarcolemma by syntrophin, and in DMD this localization is altered. Intriguingly, the presence of syntrophin on the membrane does not always restore sarcolemmal nNOS. Thus, we wished to determine whether dystrophin functions in subcellular localization of nNOS and which regions may be necessary. Using in vivo transfection of dystrophin deletion constructs, we show that sarcolemmal targeting of nNOS was dependent on the spectrin-like repeats 16 and 17 (R16/17) within the rod domain. Treatment of mdx mice (a DMD model) with R16/17-containing synthetic dystrophin genes effectively ameliorated histological muscle pathology and improved muscle strength as well as exercise performance. Furthermore, sarcolemma-targeted nNOS attenuated α-adrenergic vasoconstriction in contracting muscle and improved muscle perfusion during exercise as measured by Doppler and microsphere circulation. In summary, we have identified the dystrophin spectrin-like repeats 16 and 17 as a novel scaffold for nNOS sarcolemmal targeting. These data suggest that muscular dystrophy gene therapies based on R16/17-containing dystrophins may yield better clinical outcomes than the current therapies.
Chronic obstructive pulmonary disease (COPD) is a lethal progressive lung disease culminating in permanent airway obstruction and alveolar enlargement. Previous studies suggest CTL involvement in COPD progression; however, their precise role remains unknown. Here, we investigated whether the CTL activation receptor NK cell group 2D (NKG2D) contributes to the development of COPD. Using primary murine lung epithelium isolated from mice chronically exposed to cigarette smoke and cultured epithelial cells exposed to cigarette smoke extract in vitro, we demonstrated induced expression of the NKG2D ligand retinoic acid early transcript 1 (RAET1) as well as NKG2D-mediated cytotoxicity. Furthermore, a genetic model of inducible RAET1 expression on mouse pulmonary epithelial cells yielded a severe emphysematous phenotype characterized by epithelial apoptosis and increased CTL activation, which was reversed by blocking NKG2D activation. We also assessed whether NKG2D ligand expression corresponded with pulmonary disease in human patients by staining airway and peripheral lung tissues from never smokers, smokers with normal lung function, and current and former smokers with COPD. NKG2D ligand expression was independent of NKG2D receptor expression in COPD patients, demonstrating that ligand expression is the limiting factor in CTL activation. These results demonstrate that aberrant, persistent NKG2D ligand expression in the pulmonary epithelium contributes to the development of COPD pathologies.
Mutations in PARKIN, pten-induced putative kinase 1 (PINK1), and DJ-1 are individually linked to autosomal recessive early-onset familial forms of Parkinson disease (PD). Although mutations in these genes lead to the same disease state, the functional relationships between them and how their respective disease-associated mutations cause PD are largely unknown. Here, we show that Parkin, PINK1, and DJ-1 formed a complex (termed PPD complex) to promote ubiquitination and degradation of Parkin substrates, including Parkin itself and Synphilin-1 in neuroblastoma cells and human brain lysates. Genetic ablation of either Pink1 or Dj-1 resulted in reduced ubiquitination of endogenous Parkin as well as decreased degradation and increased accumulation of aberrantly expressed Parkin substrates. Expression of PINK1 enhanced Parkin-mediated degradation of heat shock–induced misfolded protein. In contrast, PD-pathogenic Parkin and PINK1 mutations showed reduced ability to promote degradation of Parkin substrates. This study identified a functional ubiquitin E3 ligase complex consisting of PD-associated Parkin, PINK1, and DJ-1 to promote degradation of un-/misfolded proteins and suggests that their PD-pathogenic mutations impair E3 ligase activity of the complex, which may constitute a mechanism underlying PD pathogenesis.
siRNAs that specifically silence the expression of cancer-related genes offer a therapeutic approach in oncology. However, it remains critical to determine the true mechanism of their therapeutic effects. Here, we describe the preclinical development of chemically modified siRNA targeting the essential cell-cycle proteins polo-like kinase 1 (PLK1) and kinesin spindle protein (KSP) in mice. siRNA formulated in stable nucleic acid lipid particles (SNALP) displayed potent antitumor efficacy in both hepatic and subcutaneous tumor models. This was correlated with target gene silencing following a single intravenous administration that was sufficient to cause extensive mitotic disruption and tumor cell apoptosis. Our siRNA formulations induced no measurable immune response, minimizing the potential for nonspecific effects. Additionally, RNAi-specific mRNA cleavage products were found in tumor cells, and their presence correlated with the duration of target mRNA silencing. Histological biomarkers confirmed that RNAi-mediated gene silencing effectively inhibited the target’s biological activity. This report supports an RNAi-mediated mechanism of action for siRNA antitumor effects, suggesting a new methodology for targeting other key genes in cancer development with siRNA-based therapeutics.
Copyright © 2014 American Society for Clinical Investigation