Fruit fly Drosophila melanogaster. Quorum sensing enables bacteria to communicate within and among species, and the molecule 3OC12-HSL is thought to play a central role in this system. Stoltz and colleagues investigated the role of 3OC12-HSL and one of the human enzymes that degrade it, PON1, in Pseudomonas aeruginosa virulence in the fruit fly — a tractable model for investigating the host-pathogen interaction as it lacks PONs. The authors show that human PON1 expression protected the flies from P. aeruginosa
lethality by disrupting 3OC12-HSL–mediated quorum-sensing pathways used by this pathogen.
Image credit: Photo Researchers Inc.
In the midst of the monumentally important 2008 presidential election, information about the candidates’ proposals for biomedical research is notably absent. Why is this the case, and more importantly, what can be done about it?
Laurence A. Turka
Insulin resistance is a major metabolic feature of obesity and is a key factor in the etiology of a number of diseases, including type 2 diabetes. In this review, we discuss potential mechanisms by which brief nutrient excess and obesity lead to insulin resistance and propose that these mechanisms of action are different but interrelated. We discuss how pathways that “sense” nutrients within skeletal muscle are readily able to regulate insulin action. We then discuss how obesity leads to insulin resistance via a complex interplay among systemic fatty acid excess, microhypoxia in adipose tissue, ER stress, and inflammation. In particular, we focus on the hypothesis that the macrophage is an important cell type in the propagation of inflammation and induction of insulin resistance in obesity. Overall, we provide our integrative perspective regarding how nutrients and obesity interact to regulate insulin sensitivity.
Simon Schenk, Maziyar Saberi, Jerrold M. Olefsky
Mammalian target of rapamycin (mTOR) is a component of a signaling pathway (PTEN/PI3K/AKT) that is frequently dysregulated in cancer. However, its precise relationship to the MAPK cascade (Ras/Raf/MEK/ERK), another pathway often implicated in tumorigenesis, has not been well defined. Recent evidence from tissue specimens obtained from patients who have received mTOR inhibitors suggests that ERK may be activated in response to mTOR interruption. In this issue of the JCI, Waugh Kinkade et al. and Carracedo et al. examine the relationship between these pathways in prostate and breast cancer cell model systems (see the related articles beginning on pages 3051 and 3065, respectively). Their findings suggest a link between inhibition of mTOR and ERK activation, possibly reflecting interruption of a novel negative S6K1-dependent feedback loop. Significantly, both groups observed that simultaneous inhibition of MEK/ERK and mTOR resulted in substantially enhanced antitumor effects both in vitro and in vivo. Together, these findings suggest that concurrent interruption of complementary signaling pathways warrants further investigation in cancer therapy.
Physiologic hemostasis upon injury involves many plasma proteins in a well-regulated cascade of proteolytic reactions to form a clot. Deficiency of blood coagulation Factors VIII, IX, or XI is associated with hemophilia. Factor XII (FXII) autoactivates by contact with a variety of artificial or biologic negatively charged surfaces (contact activation), resulting in blood coagulation and activation of the inflammatory kallikrein-kinin and complement systems. However, surprisingly, individuals deficient in FXII rarely suffer from bleeding disorders. Most biologic surfaces that activate FXII become expressed in disease states. Investigators have long searched for physiologic activators of FXII and its role in vivo. In this issue of the JCI, Maas et al. show that misfolded protein aggregates produced during systemic amyloidosis allow for plasma FXIIa and prekallikrein activation and increased formation of kallikrein–C1 inhibitor complexes, without Factor XIa activation and coagulation (see the related article beginning on page 3208). This study describes a novel biologic surface for FXII activation and activity, which initiates inflammatory events independent of hemostasis.
Alvin H. Schmaier
The interaction of circulating platelets with the vessel wall involves a process of cell catch and release, regulating cell rolling, skipping, or firm adhesion and leading to thrombus formation in flowing blood. In this regard, the interaction of platelet glycoprotein Ibα (GPIbα) with its adhesive ligand, vWF, is activated by shear force and critical for platelet adhesion to the vessel wall. In this issue of the JCI, Yago and colleagues show how gain-of-function mutations in the GPIbα-binding vWF A1 domain disrupt intramolecular interactions within WT vWF A1 that regulate binding to GPIbα and flow-enhanced platelet rolling and adhesion (see the related article beginning on page 3195). Together, these studies reveal molecular mechanisms regulating GPIbα-vWF bond formation and platelet adhesion under shear stress.
Robert K. Andrews, Michael C. Berndt
Inflammation plays a critical role in the development of cardiovascular diseases. Infiltration of leukocytes to sites of injury requires their exit from the blood and migration across basement membrane; this process has been postulated to require remodeling of the ECM. Plasminogen (Plg) is a protease that binds to the ECM and, upon conversion to plasmin, degrades multiple ECM proteins. In addition, plasmin directly activates MMPs. Here, we used Plg–/– mice to investigate the role of Plg in inflammatory leukocyte migration. After induction of peritonitis by thioglycollate injection, we found that Plg–/– mice displayed diminished macrophage trans-ECM migration and decreased MMP-9 activation. Furthermore, injection of the active form of MMP-9 in Plg–/– mice rescued macrophage migration in this model. We used periaortic application of CaCl2 to induce abdominal aortic aneurysm (AAA) and found that Plg–/– mice displayed reduced macrophage infiltration and were protected from aneurysm formation. Administration of active MMP-9 to Plg–/– mice promoted macrophage infiltration and the development of AAA. These data suggest that Plg regulates macrophage migration in inflammation via activation of MMP-9, which, in turn, regulates the ability of the cells to migrate across ECM. Thus, targeting the Plg/MMP-9 pathway may be an attractive approach to regulate inflammatory responses and AAA development.
Yanqing Gong, Erika Hart, Aleksey Shchurin, Jane Hoover-Plow
Atherosclerosis remains a major cause of death in the developed world despite the success of therapies that lower cholesterol and BP. The intermediate-conductance calcium-activated potassium channel KCa3.1 is expressed in multiple cell types implicated in atherogenesis, and pharmacological blockade of this channel inhibits VSMC and lymphocyte activation in rats and mice. We found that coronary vessels from patients with coronary artery disease expressed elevated levels of KCa3.1. In Apoe–/– mice, a genetic model of atherosclerosis, KCa3.1 expression was elevated in the VSMCs, macrophages, and T lymphocytes that infiltrated atherosclerotic lesions. Selective pharmacological blockade and gene silencing of KCa3.1 suppressed proliferation, migration, and oxidative stress of human VSMCs. Furthermore, VSMC proliferation and macrophage activation were reduced in KCa3.1–/– mice. In vivo therapy with 2 KCa3.1 blockers, TRAM-34 and clotrimazole, significantly reduced the development of atherosclerosis in aortas of Apoe–/– mice by suppressing VSMC proliferation and migration into plaques, decreasing infiltration of plaques by macrophages and T lymphocytes, and reducing oxidative stress. Therapeutic concentrations of TRAM-34 in mice caused no discernible toxicity after repeated dosing and did not compromise the immune response to influenza virus. These data suggest that KCa3.1 blockers represent a promising therapeutic strategy for atherosclerosis.
Kazuyoshi Toyama ... David R. Harder, Hiroto Miura
Some cases of pre–B cell acute lymphoblastic leukemia (pre–B-ALL) are caused by the Philadelphia (Ph) chromosome–encoded BCR-ABL oncogene, and these tend to have a poor prognosis. Inhibitors of the PI3K/AKT pathway reduce BCR-ABL–mediated transformation in vitro; however, the specific PI3K isoforms involved are poorly defined. Using a murine model of Ph+ pre–B-ALL, we found that deletion of both Pik3r1 and Pik3r2, genes encoding class IA PI3K regulatory isoforms, severely impaired transformation. BCR-ABL–dependent pre/pro-B cell lines could be established at low frequency from progenitors that lacked these genes, but the cells were smaller, proliferated more slowly, and failed to cause leukemia in vivo. These cell lines displayed nearly undetectable PI3K signaling function and were resistant to the PI3K inhibitor wortmannin. However, they maintained activation of mammalian target of rapamycin (mTOR) and were more sensitive to rapamycin. Treatment with rapamycin caused feedback activation of AKT in WT cell lines but not PI3K-deficient lines. A dual inhibitor of PI3K and mTOR, PI-103, was more effective than rapamycin at suppressing proliferation of mouse pre–B-ALL and human CD19+CD34+ Ph+ ALL leukemia cells treated with the ABL kinase inhibitor imatinib. Our findings provide mechanistic insights into PI3K dependency in oncogenic networks and provide a rationale for targeting class IA PI3K, alone or together with mTOR, in the treatment of Ph+ ALL.
Michael G. Kharas ... Kevan M. Shokat, David A. Fruman
The AKT/mammalian target of rapamycin (AKT/mTOR) and ERK MAPK signaling pathways have been shown to cooperate in prostate cancer progression and the transition to androgen-independent disease. We have now tested the effects of combinatorial inhibition of these pathways on prostate tumorigenicity by performing preclinical studies using a genetically engineered mouse model of prostate cancer. We report here that combination therapy using rapamycin, an inhibitor of mTOR, and PD0325901, an inhibitor of MAPK kinase 1 (MEK; the kinase directly upstream of ERK), inhibited cell growth in cultured prostate cancer cell lines and tumor growth particularly for androgen-independent prostate tumors in the mouse model. We further showed that such inhibition leads to inhibition of proliferation and upregulated expression of the apoptotic regulator Bcl-2–interacting mediator of cell death (Bim). Furthermore, analyses of human prostate cancer tissue microarrays demonstrated that AKT/mTOR and ERK MAPK signaling pathways are often coordinately deregulated during prostate cancer progression in humans. We therefore propose that combination therapy targeting AKT/mTOR and ERK MAPK signaling pathways may be an effective treatment for patients with advanced prostate cancer, in particular those with hormone-refractory disease.
Carolyn Waugh Kinkade ... Carlos Cordon-Cardo, Cory Abate-Shen
Numerous studies have established a causal link between aberrant mammalian target of rapamycin (mTOR) activation and tumorigenesis, indicating that mTOR inhibition may have therapeutic potential. In this study, we show that rapamycin and its analogs activate the MAPK pathway in human cancer, in what represents a novel mTORC1-MAPK feedback loop. We found that tumor samples from patients with biopsy-accessible solid tumors of advanced disease treated with RAD001, a rapamycin derivative, showed an administration schedule–dependent increase in activation of the MAPK pathway. RAD001 treatment also led to MAPK activation in a mouse model of prostate cancer. We further show that rapamycin-induced MAPK activation occurs in both normal cells and cancer cells lines and that this feedback loop depends on an S6K-PI3K-Ras pathway. Significantly, pharmacological inhibition of the MAPK pathway enhanced the antitumoral effect of mTORC1 inhibition by rapamycin in cancer cells in vitro and in a xenograft mouse model. Taken together, our findings identify MAPK activation as a consequence of mTORC1 inhibition and underscore the potential of a combined therapeutic approach with mTORC1 and MAPK inhibitors, currently employed as single agents in the clinic, for the treatment of human cancers.
Arkaitz Carracedo ... Jose Baselga, Pier Paolo Pandolfi
Neuronal ceroid lipofuscinoses represent the most common childhood neurodegenerative storage disorders. Infantile neuronal ceroid lipofuscinosis (INCL) is caused by palmitoyl protein thioesterase-1 (PPT1) deficiency. Although INCL patients show signs of abnormal neurotransmission, manifested by myoclonus and seizures, the molecular mechanisms by which PPT1 deficiency causes this abnormality remain obscure. Neurotransmission relies on repeated cycles of exo- and endocytosis of the synaptic vesicles (SVs), in which several palmitoylated proteins play critical roles. These proteins facilitate membrane fusion, which is required for neurotransmitter exocytosis, recycling of the fused SV membrane components, and regeneration of fresh vesicles. However, palmitoylated proteins require depalmitoylation for recycling. Using postmortem brain tissues from an INCL patient and tissue from the PPT1-knockout (PPT1-KO) mice that mimic INCL, we report here that PPT1 deficiency caused persistent membrane anchorage of the palmitoylated SV proteins, which hindered the recycling of the vesicle components that normally fuse with the presynaptic plasma membrane during SV exocytosis. Thus, the regeneration of fresh SVs, essential for maintaining the SV pool size at the synapses, was impaired, leading to a progressive loss of readily releasable SVs and abnormal neurotransmission. This abnormality may contribute to INCL neuropathology.
Sung-Jo Kim ... Louis Dye, Anil B. Mukherjee
Parkinson disease (PD) is characterized by dopaminergic neurodegeneration and intracellular inclusions of α-synuclein amyloid fibers, which are stable and difficult to dissolve. Whether inclusions are neuroprotective or pathological remains controversial, because prefibrillar oligomers may be more toxic than amyloid inclusions. Thus, whether therapies should target inclusions, preamyloid oligomers, or both is a critically important issue. In yeast, the protein-remodeling factor Hsp104 cooperates with Hsp70 and Hsp40 to dissolve and reactivate aggregated proteins. Metazoans, however, have no Hsp104 ortholog. Here we introduced Hsp104 into a rat PD model. Remarkably, Hsp104 reduced formation of phosphorylated α-synuclein inclusions and prevented nigrostriatal dopaminergic neurodegeneration induced by PD-linked α-synuclein (A30P). An in vitro assay employing pure proteins revealed that Hsp104 prevented fibrillization of α-synuclein and PD-linked variants (A30P, A53T, E46K). Hsp104 coupled ATP hydrolysis to the disassembly of preamyloid oligomers and amyloid fibers composed of α-synuclein. Furthermore, the mammalian Hsp70 and Hsp40 chaperones, Hsc70 and Hdj2, enhanced α-synuclein fiber disassembly by Hsp104. Hsp104 likely protects dopaminergic neurons by antagonizing toxic α-synuclein assemblies and might have therapeutic potential for PD and other neurodegenerative amyloidoses.
Christophe Lo Bianco ... Susan Lindquist, Patrick Aebischer
The Sox9 transcription factor plays an essential role in promoting chondrogenesis and regulating expression of chondrocyte extracellular-matrix genes. To identify genes that interact with Sox9 in promoting chondrocyte differentiation, we screened a cDNA library generated from the murine chondrogenic ATDC5 cell line to identify activators of the collagen, type II, α 1 (Col2a1) promoter. Here we have shown that paraspeckle regulatory protein 54-kDa nuclear RNA-binding protein (p54nrb) is an essential link between Sox9-regulated transcription and maturation of Sox9-target gene mRNA. We found that p54nrb physically interacted with Sox9 and enhanced Sox9-dependent transcriptional activation of the Col2a1 promoter. In ATDC5 cells, p54nrb colocalized with Sox9 protein in nuclear paraspeckle bodies, and knockdown of p54nrb suppressed Sox9-dependent Col2a1 expression and promoter activity. We generated a p54nrb mutant construct lacking RNA recognition motifs, and overexpression of mutant p54nrb in ATDC5 cells markedly altered the appearance of paraspeckle bodies and inhibited the maturation of Col2a1 mRNA. The mutant p54nrb inhibited chondrocyte differentiation of mesenchymal cells and mouse metatarsal explants. Furthermore, transgenic mice expressing the mutant p54nrb in the chondrocyte lineage exhibited dwarfism associated with impairment of chondrogenesis. These data suggest that p54nrb plays an important role in the regulation of Sox9 function and the formation of paraspeckle bodies during chondrogenesis.
Kenji Hata ... Vincent R. Harley, Toshiyuki Yoneda
Multiple receptor tyrosine kinases (RTKs), including PDGFR, have been validated as therapeutic targets in glioblastoma multiforme (GBM), yet inhibitors of RTKs have had limited clinical success. As various antiapoptotic mechanisms render GBM cells resistant to chemo- and radiotherapy, we hypothesized that these antiapoptotic mechanisms also confer resistance to RTK inhibition. We found that in vitro inhibition of PDGFR in human GBM cells initiated the intrinsic pathway of apoptosis, as evidenced by mitochondrial outer membrane permeabilization, but downstream caspase activation was blocked by inhibitor of apoptosis proteins (IAPs). Consistent with this, inhibition of PDGFR combined with small molecule inactivation of IAPs induced apoptosis in human GBM cells in vitro and had synergistic antitumor effects in orthotopic mouse models of GBM and in primary human GBM neurospheres. These results demonstrate that concomitant inhibition of IAPs can overcome resistance to RTK inhibitors in human malignant GBM cells, and suggest that blockade of IAPs has the potential to improve treatment outcomes in patients with GBM.
David S. Ziegler ... Leigh Zawel, Andrew L. Kung
Pseudomonas aeruginosa uses quorum sensing, an interbacterial communication system, to regulate gene expression. The signaling molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL) is thought to play a central role in quorum sensing. Since 3OC12-HSL can be degraded by paraoxonase (PON) family members, we hypothesized that PONs regulate P. aeruginosa virulence in vivo. We chose Drosophila melanogaster as our model organism because it has been shown to be a tractable model for investigating host-pathogen interactions and lacks PONs. By using quorum-sensing–deficient P. aeruginosa, synthetic acyl-HSLs, and transgenic expression of human PON1, we investigated the role of 3OC12-HSL and PON1 on P. aeruginosa virulence. We found that P. aeruginosa virulence in flies was dependent upon 3OC12-HSL. PON1 transgenic flies expressed enzymatically active PON1 and thereby exhibited arylesterase activity and resistance to organophosphate toxicity. Moreover, PON1 flies were protected from P. aeruginosa lethality, and protection was dependent on the lactonase activity of PON1. Our findings show that PON1 can interfere with quorum sensing in vivo and provide insight into what we believe is a novel role for PON1 in the innate immune response to quorum-sensing–dependent pathogens. These results raise intriguing possibilities about human-pathogen interactions, including potential roles for PON1 as a modifier gene and for PON1 protein as a regulator of normal bacterial florae, a link between infection/inflammation and cardiovascular disease, and a potential therapeutic modality.
David A. Stoltz ... Matthew R. Parsek, Joseph Zabner
Previously, several individuals with X-linked SCID (SCID-X1) were treated by gene therapy to restore the missing IL-2 receptor γ (IL2RG) gene to CD34+ BM precursor cells using gammaretroviral vectors. While 9 of 10 patients were successfully treated, 4 of the 9 developed T cell leukemia 31–68 months after gene therapy. In 2 of these cases, blast cells contained activating vector insertions near the LIM domain–only 2 (LMO2) proto-oncogene. Here, we report data on the 2 most recent adverse events, which occurred in patients 7 and 10. In patient 10, blast cells contained an integrated vector near LMO2 and a second integrated vector near the proto-oncogene BMI1. In patient 7, blast cells contained an integrated vector near a third proto-oncogene,CCND2. Additional genetic abnormalities in the patients’ blast cells included chromosomal translocations, gain-of-function mutations activating NOTCH1, and copy number changes, including deletion of tumor suppressor gene CDKN2A, 6q interstitial losses, and SIL-TAL1 rearrangement. These findings functionally specify a genetic network that controls growth in T cell progenitors. Chemotherapy led to sustained remission in 3 of the 4 cases of T cell leukemia, but failed in the fourth. Successful chemotherapy was associated with restoration of polyclonal transduced T cell populations. As a result, the treated patients continued to benefit from therapeutic gene transfer.
Salima Hacein-Bey-Abina ... Alain Fischer, Marina Cavazzana-Calvo
X-linked SCID (SCID-X1) is amenable to correction by gene therapy using conventional gammaretroviral vectors. Here, we describe the occurrence of clonal T cell acute lymphoblastic leukemia (T-ALL) promoted by insertional mutagenesis in a completed gene therapy trial of 10 SCID-X1 patients. Integration of the vector in an antisense orientation 35 kb upstream of the protooncogene LIM domain only 2 (LMO2) caused overexpression of LMO2 in the leukemic clone. However, leukemogenesis was likely precipitated by the acquisition of other genetic abnormalities unrelated to vector insertion, including a gain-of-function mutation in NOTCH1, deletion of the tumor suppressor gene locus cyclin-dependent kinase 2A (CDKN2A), and translocation of the TCR-β region to the STIL-TAL1 locus. These findings highlight a general toxicity of endogenous gammaretroviral enhancer elements and also identify a combinatorial process during leukemic evolution that will be important for risk stratification and for future protocol design.
Steven J. Howe ... H. Bobby Gaspar, Adrian J. Thrasher
PKC isoforms t, α, and β play fundamental roles in the activation of T cells and other immune cell functions. Here we show that the PKC inhibitor AEB071 both abolishes the production of several cytokines by activated human T cells, keratinocytes, and macrophages in vitro and inhibits an acute allergic contact dermatitis response in rats. To translate these findings into humans, single and multiple ascending oral doses of AEB071 were administered to healthy volunteers and patients with psoriasis, respectively. AEB071 was well tolerated with no clinically relevant laboratory abnormalities. Ex vivo stimulation of lymphocytes from subjects exposed to single doses of AEB071 resulted in a dose-dependent inhibition of both lymphocyte proliferation and IL2 mRNA expression. Clinical severity of psoriasis was reduced up to 69% compared with baseline after 2 weeks of treatment, as measured by the Psoriasis Area Severity Index (PASI) score. The improvement in psoriasis patients was accompanied by histological improvement of skin lesions and may be partially explained by a substantial reduction of p40+ dermal cells, which are known to mediate psoriasis. These data suggest that AEB071 could be an effective novel treatment regimen for psoriasis and other autoimmune diseases, and that AEB071 warrants long-term studies to establish safety and efficacy.
Hans Skvara ... Georg Stingl, Thomas Jung
Diet-induced obesity is associated with fatty liver, insulin resistance, leptin resistance, and changes in plasma lipid profile. Endocannabinoids have been implicated in the development of these associated phenotypes, because mice deficient for the cannabinoid receptor CB1 (CB1–/–) do not display these changes in association with diet-induced obesity. The target tissues that mediate these effects, however, remain unknown. We therefore investigated the relative role of hepatic versus extrahepatic CB1 receptors in the metabolic consequences of a high-fat diet, using liver-specific CB1 knockout (LCB1–/–) mice. LCB1–/– mice fed a high-fat diet developed a similar degree of obesity as that of wild-type mice, but, similar to CB1–/– mice, had less steatosis, hyperglycemia, dyslipidemia, and insulin and leptin resistance than did wild-type mice fed a high-fat diet. CB1 agonist–induced increase in de novo hepatic lipogenesis and decrease in the activity of carnitine palmitoyltransferase–1 and total energy expenditure were absent in both CB1–/– and LCB1–/– mice. We conclude that endocannabinoid activation of hepatic CB1 receptors contributes to the diet-induced steatosis and associated hormonal and metabolic changes, but not to the increase in adiposity, observed with high-fat diet feeding. Theses studies suggest that peripheral CB1 receptors could be selectively targeted for the treatment of fatty liver, impaired glucose homeostasis, and dyslipidemia in order to minimize the neuropsychiatric side effects of nonselective CB1 blockade during treatment of obesity-associated conditions.
Douglas Osei-Hyiaman ... Christoph Buettner, George Kunos
Iatrogenic tumor cell implantation within surgical wounds can compromise curative cancer surgery. Adhesion of cancer cells, in particular colon cancer cells, is stimulated by exposure to increased extracellular pressure through a cytoskeleton-dependent signaling mechanism requiring FAK, Src, Akt, and paxillin. Mechanical stimuli during tumor resection may therefore negatively impact patient outcome. We hypothesized that perioperative administration of colchicine, which prevents microtubule polymerization, could disrupt pressure-stimulated tumor cell adhesion to surgical wounds and enhance tumor-free survival. Ex vivo treatment of Co26 and Co51 colon cancer cells with colchicine inhibited pressure-stimulated cell adhesion to murine surgical wounds and blocked pressure-induced FAK and Akt phosphorylation. Surgical wound contamination with pressure-activated Co26 and Co51 cells significantly reduced tumor-free survival compared with contamination with tumor cells under ambient pressure. Mice treated with pressure-activated Co26 and Co51 cells from tumors preoperatively treated with colchicine in vivo displayed reduced surgical site implantation and significantly increased tumor-free survival compared with mice exposed to pressure-activated cells from tumors not pretreated with colchicine. Our data suggest that pressure activation of malignant cells promotes tumor development and impairs tumor-free survival and that perioperative colchicine administration or similar interventions may inhibit this effect.
David H. Craig, Cheri R. Owen, William C. Conway, Mary F. Walsh, Christina Downey, Marc D. Basson
Gain-of-function NOTCH1 mutations are found in 50%–70% of human T cell acute lymphoblastic leukemia/lymphoma (T-ALL) cases. Gain-of-function NOTCH1 alleles that initiate strong downstream signals induce leukemia in mice, but it is unknown whether the gain-of-function NOTCH1 mutations most commonly found in individuals with T-ALL generate downstream signals of sufficient strength to induce leukemia. We addressed this question by expressing human gain-of-function NOTCH1 alleles of varying strength in mouse hematopoietic precursors. Uncommon gain-of-function NOTCH1 alleles that initiated strong downstream signals drove ectopic T cell development and induced leukemia efficiently. In contrast, although gain-of-function alleles that initiated only weak downstream signals also induced ectopic T cell development, these more common alleles failed to efficiently initiate leukemia development. However, weak gain-of-function NOTCH1 alleles accelerated the onset of leukemia initiated by constitutively active K-ras and gave rise to tumors that were sensitive to Notch signaling pathway inhibition. These data show that induction of leukemia requires doses of Notch1 greater than those needed for T cell development and that most NOTCH1 mutations found in T-ALL cells do not generate signals of sufficient strength to initiate leukemia development. Furthermore, low, nonleukemogenic levels of Notch1 can complement other leukemogenic events, such as activation of K-ras. Even when Notch1 participates secondarily, the resulting tumors show “addiction” to Notch, providing a further rationale for evaluating Notch signaling pathway inhibitors in leukemia.
Mark Y. Chiang ... Jon C. Aster, Warren S. Pear
Arterial blood flow enhances glycoprotein Ibα (GPIbα) binding to vWF, which initiates platelet adhesion to injured vessels. Mutations in the vWF A1 domain that cause type 2B von Willebrand disease (vWD) reduce the flow requirement for adhesion. Here we show that increasing force on GPIbα/vWF bonds first prolonged (“catch”) and then shortened (“slip”) bond lifetimes. Two type 2B vWD A1 domain mutants, R1306Q and R1450E, converted catch bonds to slip bonds by prolonging bond lifetimes at low forces. Steered molecular dynamics simulations of GPIbα dissociating from the A1 domain suggested mechanisms for catch bonds and their conversion by the A1 domain mutations. Catch bonds caused platelets and GPIbα-coated microspheres to roll more slowly on WT vWF and WT A1 domains as flow increased from suboptimal levels, explaining flow-enhanced rolling. Longer bond lifetimes at low forces eliminated the flow requirement for rolling on R1306Q and R1450E mutant A1 domains. Flowing platelets agglutinated with microspheres bearing R1306Q or R1450E mutant A1 domains, but not WT A1 domains. Therefore, catch bonds may prevent vWF multimers from agglutinating platelets. A disintegrin and metalloproteinase with a thrombospondin type 1 motif–13 (ADAMTS-13) reduced platelet agglutination with microspheres bearing a tridomain A1A2A3 vWF fragment with the R1450E mutation in a shear-dependent manner. We conclude that in type 2B vWD, prolonged lifetimes of vWF bonds with GPIbα on circulating platelets may allow ADAMTS-13 to deplete large vWF multimers, causing bleeding.
Tadayuki Yago ... Rodger P. McEver, Cheng Zhu
When blood is exposed to negatively charged surface materials such as glass, an enzymatic cascade known as the contact system becomes activated. This cascade is initiated by autoactivation of Factor XII and leads to both coagulation (via Factor XI) and an inflammatory response (via the kallikrein-kinin system). However, while Factor XII is important for coagulation in vitro, it is not important for physiological hemostasis, so the physiological role of the contact system remains elusive. Using patient blood samples and isolated proteins, we identified a novel class of Factor XII activators. Factor XII was activated by misfolded protein aggregates that formed by denaturation or by surface adsorption, which specifically led to the activation of the kallikrein-kinin system without inducing coagulation. Consistent with this, we found that Factor XII, but not Factor XI, was activated and kallikrein was formed in blood from patients with systemic amyloidosis, a disease marked by the accumulation and deposition of misfolded plasma proteins. These results show that the kallikrein-kinin system can be activated by Factor XII, in a process separate from the coagulation cascade, and point to a protective role for Factor XII following activation by misfolded protein aggregates.
Coen Maas ... Bonno N. Bouma, Martijn F.B.G. Gebbink
β3-adrenergic receptor (β3-AR) activation produces a negative inotropic effect in human ventricles. Here we explored the role of β3-AR in the human atrium. Unexpectedly, β3-AR activation increased human atrial tissue contractility and stimulated the L-type Ca2+ channel current (ICa,L) in isolated human atrial myocytes (HAMs). Right atrial tissue specimens were obtained from 57 patients undergoing heart surgery for congenital defects, coronary artery diseases, valve replacement, or heart transplantation. The ICa,L and isometric contraction were recorded using a whole-cell patch-clamp technique and a mechanoelectrical force transducer. Two selective β3-AR agonists, SR58611 and BRL37344, and a β3-AR partial agonist, CGP12177, stimulated ICa,L in HAMs with nanomolar potency and a 60%–90% efficacy compared with isoprenaline. The β3-AR agonists also increased contractility but with a much lower efficacy (~10%) than isoprenaline. The β3-AR antagonist L-748,337, β1-/β2-AR antagonist nadolol, and β1-/β2-/β3-AR antagonist bupranolol were used to confirm the involvement of β3-ARs (and not β1-/β2-ARs) in these effects. The β3-AR effects involved the cAMP/PKA pathway, since the PKA inhibitor H89 blocked ICa,L stimulation and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) strongly increased the positive inotropic effect. Therefore, unlike in ventricular tissue, β3-ARs are positively coupled to L-type Ca2+ channels and contractility in human atrial tissues through a cAMP-dependent pathway.
V. Arvydas Skeberdis ... Jonas Jurevičius, Rodolphe Fischmeister
The pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) are closely related orphan nuclear hormone receptors that play a critical role as xenobiotic sensors in mammals. Both receptors regulate the expression of genes involved in the biotransformation of chemicals in a ligand-dependent manner. As the ligand specificity of PXR and CAR have diverged between species, the prediction of in vivo PXR and CAR interactions with a drug are difficult to extrapolate from animals to humans. We report the development of what we believe are novel PXR- and CAR-humanized mice, generated using a knockin strategy, and Pxr- and Car-KO mice as well as a panel of mice including all possible combinations of these genetic alterations. The expression of human CAR and PXR was in the predicted tissues at physiological levels, and splice variants of both human receptors were expressed. The panel of mice will allow the dissection of the crosstalk between PXR and CAR in the response to different drugs. To demonstrate the utility of this panel of mice, we used the mice to show that the in vivo induction of Cyp3a11 and Cyp2b10 by phenobarbital was only mediated by CAR, although this compound is described as a PXR and CAR activator in vitro. This panel of mouse models is a useful tool to evaluate the roles of CAR and PXR in drug bioavailability, toxicity, and efficacy in humans.
Nico Scheer, Jillian Ross, Anja Rode, Branko Zevnik, Sandra Niehaves, Nicole Faust, C. Roland Wolf
Copyright © 2014 American Society for Clinical Investigation