Volume 115, Issue 4, Pages 789-1100
47 total articles
The recent uproar over comments about women in science made by Harvard University President Dr. Lawrence Summers has helped focus much needed attention on an issue of great importance. Whatever motivated Dr. Summers to make statements about the “different availability of aptitude at the high end” as a possible explanation for the lack of gender diversity in science and engineering, we should all thank him for putting the question of why there are not more women in science where it belongs — front and center.
Perhaps because I am a veteran of the “good old days” (they were really quite bad), young physicians who hope to become clinical investigators often ask me how they might establish their careers. Many are more than a little worried about their futures and often have trouble envisioning a career path that is financially secure for themselves and their families. The grumbling of clinical investigators a few years their senior enhances their angst. So I try to encourage these young physicians because I know the great intellectual (if not monetary) rewards of the field and because I know that the future of medicine absolutely depends on clinical investigators. The following is what I try to say to them.
Cutaneous T cell lymphomas (CTCLs) are a heterogenous group of lymphoproliferative disorders caused by clonally derived, skin-invasive T cells. Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common types of CTCLs and are characterized by malignant CD4+/CLA+/CCR4+ T cells that also lack the usual T cell surface markers CD7 and/or CD26. As MF/SS advances, the clonal dominance of the malignant cells results in the expression of predominantly Th2 cytokines, progressive immune dysregulation in patients, and further tumor cell growth. This review summarizes recent insights into the pathogenesis and immunobiology of MF/SS and how these have shaped current therapeutic approaches, in particular the growing emphasis on enhancement of host antitumor immune responses as the key to successful therapy.
Melanoma is a cancer of the neural crest–derived cells that provide pigmentation to skin and other tissues. Over the past 4 decades, the incidence of melanoma has increased more rapidly than that of any other malignancy in the United States. No current treatments substantially enhance patient survival once metastasis has occurred. This review focuses on recent insights into melanoma genetics and new therapeutic approaches being developed based on these advances.
Knowledge of the pathophysiology of immunobullous diseases has been advanced by the demonstration that passive transfer of antibodies against skin autoantigens can induce blisters in experimental animals with clinical, histologic, and immunopathologic features similar to those seen in human patients. In this issue of the JCI, Liu et al. extend their earlier observations regarding an experimental murine model of bullous pemphigoid by showing that the plasminogen/plasmin signaling cascade synergizes with MMP-9 during the early phase of antibody-induced blister formation in vivo. In a separate study, Sitaru et al. show for the first time to my knowledge that passive transfer of experimental antibodies against type VII collagen create subepidermal blisters in mice that mimic those seen in patients with epidermolysis bullosa acquisita (see the related article beginning on page 870). While the articles by Liu, Sitaru, and their colleagues identify pathways of inflammation and tissue injury that, if interrupted, may abrogate blister formation, in a third study, Payne et al. utilized phage display technologies to isolate human anti-desmoglein monoclonal antibodies from a patient with pemphigus vulgaris and show that such antibodies have restricted patterns of heavy and light chain gene usage — findings suggesting that autoantibodies may represent an additional target for therapeutic interventions in patients with immunobullous diseases (see the related article beginning on page 888).
Every cell in the body expresses a set of proteins designed to trigger permeabilization of the mitochondria and cell death. Inactivation or inappropriate triggering of these pathways is increasingly recognized as a contributor to human disease. A study in this issue of the JCI demonstrates that IL-6 exerts its protective effect against the development of lung injury following exposure of mice to 95% O2 by increasing the expression of a Bcl-2–related protein, A1. This protein acts to prevent mitochondrial membrane permeabilization and cell death following exposure to hyperoxia. The data in this study lend support to the hypothesis that inappropriate triggering of cell-death pathways may contribute to the development of hyperoxic pulmonary edema, lung injury, and respiratory failure.
Rare mutations in MEF2A have been proposed as a cause of coronary artery disease (CAD) and myocardial infarction (MI). In this issue of the JCI, Pennacchio and colleagues report sequencing MEF2A in 300 patients with premature CAD and in controls. Only 1 CAD patient was found to carry a missense mutation not found in controls. The specific 21-bp deletion in MEF2A previously proposed as causal for CAD and/or MI was observed in unaffected individuals and did not segregate with CAD in families. These results do not support the hypothesis that mutations in MEF2A are a cause of CAD and/or MI but do illustrate general principles regarding the difficulty of connecting genetic variation to common diseases.
In this issue of the JCI, Niedermaier and colleagues demonstrate that a chromosomal inversion in mice results in dysregulation of Sonic hedgehog (Shh), such that Shh is ectopically expressed in a skeletogenic domain typically occupied by Indian hedgehog (Ihh). This molecular reversal eliminates phalangeal joint spaces, and consequently, Short digits (Dsh) heterozygotes (Dsh/+) have brachydactyly (shortened digits). Ihh is normally downregulated in regions that will become the joint space, but in Dsh/+ mice, Shh bypasses this regulatory control and persists; accordingly, cells maintain their chondrogenic fate and the developed digits are shorter than normal. The significance of these data extends far beyond the field of skeletal biology: they hint at the very real possibility that the endogenous Shh regulatory region contains a repressor designed to segregate the activity of Shh from Ihh. The existence of such a repressor provides a window into the distant past, revealing that Shh and Ihh must once have shared responsibilities in establishing tissue boundaries and orchestrating vertebrate tissue morphogenesis.
In systemic lupus erythematosus (SLE), IL-2 production by T lymphocytes in vitro is impaired. Deficient IL-2 production may be an outcome of a primary SLE T cell disorder that is due to impaired signal transduction. In this issue of the JCI, evidence is presented that an anti-TCR/CD3 complex autoantibody present in SLE sera can bind to T cells and activate the Ca2+-calmodulin kinase IV (CaMKIV) signaling cascade, resulting in downregulation of IL-2 transcription and IL-2 production. Because IL-2 may contribute to the maintenance of T cell tolerance, deficient IL-2 production could promote a breach of T cell tolerance that results in autoantibody production in SLE.
Blood pressure abnormalities are thought to originate from intrinsic changes in the kidney, a concept that has been largely unchallenged for more than 4 decades. However, recent molecular, cellular, and transgenic mouse studies support an alternative hypothesis: primary abnormalities in vascular cell function can also directly cause abnormalities of blood pressure. In this issue of the JCI, Crowley and coworkers describe the application of an elegant cross-renal transplant model to type 1A angiotensin (AT1A) receptor–deficient mice and their wild-type littermates to explore the relative contributions of renal and extrarenal tissues to the low blood pressure seen in the AT1A receptor–deficient animals. Their studies further support the emerging paradigm that primary abnormalities of the vasculature can make unique, nonredundant contributions to blood pressure regulation; the findings have potentially important implications for the ways we diagnose and treat blood pressure diseases in humans.
The hypothalamic-pituitary-adrenal axis (a major component of the stress system) and the immune system contribute to the maintenance of homeostasis at rest and during stress. Because of their essential roles for the survival of self and species, the activities of these systems have evolutionarily developed in parallel and are intertwined at many levels. In this issue of the JCI, Ezzat et al. demonstrate that Ikaros, a differentiation factor of leukocyte lineage, also influences the maturation of the fetal pituitary corticotroph and, hence, the secretion of adrenocorticotropic hormone before and after birth. These results indicate that Ikaros is an ontogenetic and phylogenetic integrator of the stress and immune systems and that abnormalities in its function may produce endocrine and/or immune pathologies.
The inhibitor of NF-κB (I-κB) kinase (IKK) complex consists of 3 subunits, IKK1, IKK2, and NF-κB essential modulator (NEMO), and is involved in the activation of NF-κB by various stimuli. IKK2 or NEMO constitutive knockout mice die during embryogenesis as a result of massive hepatic apoptosis. Therefore, we examined the role of IKK2 in TNF-induced apoptosis and ischemia/reperfusion (I/R) injury in the liver by using conditional knockout mice. Hepatocyte-specific ablation of IKK2 did not lead to impaired activation of NF-κB or increased apoptosis after TNF-α stimulation whereas conditional NEMO knockout resulted in complete block of NF-κB activation and massive hepatocyte apoptosis. In a model of partial hepatic I/R injury, mice lacking IKK2 in hepatocytes displayed significantly reduced liver necrosis and inflammation than wild-type mice. AS602868, a novel chemical inhibitor of IKK2, protected mice from liver injury due to I/R without sensitizing them toward TNF-induced apoptosis and could therefore emerge as a new pharmacological therapy for liver resection, hemorrhagic shock, or transplantation surgery.
Increasing evidence demonstrates that IL-6 has a protective role during liver injury. IL-6 activates intracellular pathways via the gp130 receptor. In order to identify IL-6–gp130 pathways involved in mediating liver protection, we analyzed hepatocyte-specific gp130 knockout mice in a concanavalin A–induced (Con A–induced) model of immune-mediated hepatitis. We demonstrated that IL-6–gp130–dependent pathways in hepatocytes alone are sufficient for triggering protection in Con A–induced hepatitis. gp130-STAT3 signaling in hepatocytes mediates the IL-6–triggered protective effect. This was demonstrated by analysis of IL-6–induced protection in mice selectively deficient for gp130-dependent STAT1/3 or gp130-SHP2-RAS signaling in hepatocytes. To identify IL-6–gp130–STAT1/3 dependently expressed liver-protective factors, we performed gene array analysis of hepatic gene expression in hepatocyte-specific gp130–/– mice as well as in gp130-STAT1/3– and gp130-SHP2-RAS-MAPK–deficient mice. The mouse IL-8 ortholog KC (also known as Gro-α) and serum amyloid A2 (SAA2) was identified as differentially IL-6–gp130–STAT3–regulated genes. Hepatic expression of KC and SAA2 mediate the liver-protective potential of IL-6, since treatment with recombinant KC or serum SAA2 effectively reduced liver injury during Con A–induced hepatitis. In summary, this study defines IL-6–gp130–STAT3–dependent gene expression in hepatocytes that mediates IL-6–triggered protection in immune-mediated Con A–induced hepatitis. Additionally, we identified the IL-6–gp130–STAT3–dependent proteins KC and SAA2 as new candidates for therapeutic targets in liver diseases.
Epidermolysis bullosa acquisita (EBA) is a subepidermal blistering disorder associated with tissue-bound and circulating autoantibodies specific to type VII collagen, a major constituent of the dermal-epidermal junction. Previous attempts to transfer the disease by injection of patient autoantibodies into mice have been unsuccessful. To study the pathogenic relevance of antibodies specific to type VII collagen in vivo, we generated and characterized rabbit antibodies specific to a murine form of this antigen and passively transferred them into adult nude, BALB/c, and C57BL/6 mice. Immune rabbit IgG bound to the lamina densa of murine skin and immunoblotted type VII collagen. Mice injected with purified IgG specific to type VII collagen, in contrast to control mice, developed subepidermal skin blisters, reproducing the human disease at the clinical, histological, electron microscopical, and immunopathological levels. Titers of rabbit IgG in the serum of mice correlated with the extent of the disease. F(ab′)2 fragments of rabbit IgG specific to type VII collagen were not pathogenic. When injected into C5-deficient mice, antibodies specific to type VII collagen failed to induce the disease, whereas C5-sufficient mice were susceptible to blister induction. This animal model for EBA should facilitate further dissection of the pathogenesis of this disease and development of new therapeutic strategies.
Extracellular proteolysis by the plasminogen/plasmin (Plg/plasmin) system and MMPs is required for tissue injury in autoimmune and inflammatory diseases. We demonstrate that a Plg cascade synergizes with MMP-9/gelatinase B in vivo during dermal-epidermal separation in an experimental model of bullous pemphigoid (BP), an autoimmune disease. BP was induced in mice by antibodies to the hemidesmosomal antigen BP180. Mice deficient in MMP-9 were resistant to experimental BP, while mice deficient in Plg and both tissue Plg activator (tPA) and urokinase Plg activator (uPA) showed delayed and less intense blister formation induced by antibodies to BP180. Plg-deficient mice reconstituted locally with Plg or the active form of MMP-9 (actMMP-9), but not the proenzyme form of MMP-9 (proMMP-9), developed BP. In contrast, proMMP-9 or actMMP-9, but not Plg, reconstituted susceptibility of MMP-9–deficient mice to the skin disease. In addition, MMP-3–deficient mice injected with pathogenic IgG developed the same degree of BP and expressed levels of actMMP-9 in the skin similar to those of WT controls. Thus, the Plg/plasmin system is epistatic to MMP-9 activation and subsequent dermal-epidermal separation in BP.
Pemphigus is a life-threatening blistering disorder of the skin and mucous membranes caused by pathogenic autoantibodies to desmosomal adhesion proteins desmoglein 3 (Dsg3) and Dsg1. Mechanisms of antibody pathogenicity are difficult to characterize using polyclonal patient sera. Using antibody phage display, we have isolated repertoires of human anti-Dsg mAbs as single-chain variable-region fragments (scFvs) from a patient with active mucocutaneous pemphigus vulgaris. ScFv mAbs demonstrated binding to Dsg3 or Dsg1 alone, or both Dsg3 and Dsg1. Inhibition ELISA showed that the epitopes defined by these scFvs are blocked by autoantibodies from multiple pemphigus patients. Injection of scFvs into neonatal mice identified 2 pathogenic scFvs that caused blisters histologically similar to those observed in pemphigus patients. Similarly, these 2 scFvs, but not others, induced cell sheet dissociation of cultured human keratinocytes, indicating that both pathogenic and nonpathogenic antibodies were isolated. Genetic analysis of these mAbs showed restricted patterns of heavy and light chain gene usage, which were distinct for scFvs with different desmoglein-binding specificities. Detailed characterization of these pemphigus mAbs should lead to a better understanding of the immunopathogenesis of disease and to more specifically targeted therapeutic approaches.
Short digits (Dsh) is a radiation-induced mouse mutant. Homozygous mice are characterized by multiple defects strongly resembling those resulting from Sonic hedgehog (Shh) inactivation. Heterozygous mice show a limb reduction phenotype with fusion and shortening of the proximal and middle phalanges in all digits, similar to human brachydactyly type A1, a condition caused by mutations in Indian hedgehog (IHH). We mapped Dsh to chromosome 5 in a region containing Shh and were able to demonstrate an inversion comprising 11.7 Mb. The distal breakpoint is 13.298 kb upstream of Shh, separating the coding sequence from several putative regulatory elements identified by interspecies comparison. The inversion results in almost complete downregulation of Shh expression during E9.5–E12.5, explaining the homozygous phenotype. At E13.5 and E14.5, however, Shh is upregulated in the phalangeal anlagen of Dsh/+ mice, at a time point and in a region where WT Shh is never expressed. The dysregulation of Shh expression causes the local upregulation of hedgehog target genes such as Gli1-3, patched, and Pthlh, as well as the downregulation of Ihh and Gdf5. This results in shortening of the digits through an arrest of chondrocyte differentiation and the disruption of joint development.
Autosomal dominant polycystic kidney disease (ADPKD) is the most common human monogenic genetic disorder and is characterized by progressive bilateral renal cysts and the development of renal insufficiency. The cystogenesis of ADPKD is believed to be a monoclonal proliferation of PKD-deficient (PKD–/–) renal tubular epithelial cells. To define the function of Pkd1, we generated chimeric mice by aggregation of Pkd1–/– ES cells and Pkd1+/+ morulae from ROSA26 mice. As occurs in humans with ADPKD, these mice developed cysts in the kidney, liver, and pancreas. Surprisingly, the cyst epithelia of the kidney were composed of both Pkd1–/– and Pkd1+/+ renal tubular epithelial cells in the early stages of cystogenesis. Pkd1–/– cyst epithelial cells changed in shape from cuboidal to flat and replaced Pkd1+/+ cyst epithelial cells lost by JNK-mediated apoptosis in intermediate stages. In late-stage cysts, Pkd1–/– cells continued immortalized proliferation with downregulation of p53. These results provide a novel understanding of the cystogenesis of ADPKD patients. Furthermore, immortalized proliferation without induction of p53 was frequently observed in 3T3-type culture of mouse embryonic fibroblasts from Pkd1–/– mice. Thus, Pkd1 plays a role in preventing immortalized proliferation of renal tubular epithelial cells through the induction of p53 and activation of JNK.
The mechanisms by which mixed-lineage leukemia (MLL) fusion products resulting from in utero translocations in 11q23 contribute to leukemogenesis and infant acute leukemia remain elusive. It is still controversial whether the MLL fusion protein is sufficient to induce acute leukemia without additional genetic alterations, although carcinogenesis in general is known to result from more than 1 genetic disorder accumulating during a lifetime. Here we demonstrate that the fusion partner–mediated homo-oligomerization of MLL-SEPT6 is essential to immortalize hematopoietic progenitors in vitro. MLL-SEPT6 induced myeloproliferative disease with long latency in mice, but not acute leukemia, implying that secondary genotoxic events are required to develop leukemia. We developed in vitro and in vivo model systems of leukemogenesis by MLL fusion proteins, where activated FMS-like receptor tyrosine kinase 3 (FLT3) together with MLL-SEPT6 not only transformed hematopoietic progenitors in vitro but also induced acute biphenotypic or myeloid leukemia with short latency in vivo. In these systems, MLL-ENL, another type of the fusion product that seems to act as a monomer, also induced the transformation in vitro and leukemogenesis in vivo in concert with activated FLT3. These findings show direct evidence for a multistep leukemogenesis mediated by MLL fusion proteins and may be applicable to development of direct MLL fusion–targeted therapy.
Homeostatic regulation of T cells involves an ongoing balance of new T cell generation, peripheral expansion, and turnover. The recovery of T cells when this balance is disrupted provides insight into the mechanisms that govern homeostasis. In a long-term, single cohort study, we assessed the role of thymic function after autologous transplant in adults, correlating serial computed tomography imaging of thymic size with concurrent measurements of peripheral CD4+ T cell populations. We established the age-dependent incidence, time course, and duration of thymic enlargement in adults and demonstrated that these changes were correlated with peripheral recovery of naive CD45RA+CD62L+ and signal-joint TCR rearrangement excision circle–bearing CD4+ populations with broad TCR diversity. Furthermore, we demonstrated that renewed thymopoiesis was critical for the restoration of peripheral CD4+ T cell populations. This recovery encompassed the recovery of normal CD4+ T cell numbers, a low ratio of effector to central memory cells, and a broad repertoire of TCR Vβ diversity among these memory cells. These data define the timeline and consequences of renewal of adult thymopoietic activity at levels able to quantitatively restore peripheral T cell populations. They further suggest that structural thymic regrowth serves as a basis for the regeneration of peripheral T cell populations.
Insulin receptor substrate 2 (Irs2) plays complex roles in energy homeostasis. We generated mice lacking Irs2 in β cells and a population of hypothalamic neurons (RIPCreIrs2KO), in all neurons (NesCreIrs2KO), and in proopiomelanocortin neurons (POMCCreIrs2KO) to determine the role of Irs2 in the CNS and β cell. RIPCreIrs2KO mice displayed impaired glucose tolerance and reduced β cell mass. Overt diabetes did not ensue, because β cells escaping Cre-mediated recombination progressively populated islets. RIPCreIrs2KO and NesCreIrs2KO mice displayed hyperphagia, obesity, and increased body length, which suggests altered melanocortin action. POMCCreIrs2KO mice did not display this phenotype. RIPCreIrs2KO and NesCreIrs2KO mice retained leptin sensitivity, which suggests that CNS Irs2 pathways are not required for leptin action. NesCreIrs2KO and POMCCreIrs2KO mice did not display reduced β cell mass, but NesCreIrs2KO mice displayed mild abnormalities of glucose homeostasis. RIPCre neurons did not express POMC or neuropeptide Y. Insulin and a melanocortin agonist depolarized RIPCre neurons, whereas leptin was ineffective. Insulin hyperpolarized and leptin depolarized POMC neurons. Our findings demonstrate a critical role for IRS2 in β cell and hypothalamic function and provide insights into the role of RIPCre neurons, a distinct hypothalamic neuronal population, in growth and energy homeostasis.
Central control of energy balance depends on the ability of proopiomelanocortin (POMC) or agouti-related protein (Agrp) hypothalamic neurons to sense and respond to changes in peripheral energy stores. Leptin and insulin have been implicated as circulating indicators of adiposity, but it is not clear how changes in their levels are perceived or integrated by individual neuronal subtypes. We developed mice in which a fluorescent reporter for PI3K activity is targeted to either Agrp or POMC neurons and used 2-photon microscopy to measure dynamic regulation of PI3K by insulin and leptin in brain slices. We show that leptin and insulin act in parallel to stimulate PI3K in POMC neurons but in opposite ways on Agrp neurons. These results suggest a new view of hypothalamic circuitry, in which the effects of leptin and insulin are integrated by anorexigenic but not by orexigenic neurons.
Lipid rafts are cholesterol- and sphingolipid-enriched microdomains in cell membranes that regulate phosphorylation cascades originating from membrane-bound proteins. In this study, we tested whether alteration of the cholesterol content of lipid rafts in prostate cancer (PCa) cell membranes affects cell survival mechanisms in vitro and in vivo. Simvastatin, a cholesterol synthesis inhibitor, lowered raft cholesterol content, inhibited Akt1 serine-threonine kinase (protein kinase Bα)/protein kinase B (Akt/PKB) pathway signaling, and induced apoptosis in caveolin- and PTEN-negative LNCaP PCa cells. Replenishing cell membranes with cholesterol reversed these inhibitory and apoptotic effects. Cholesterol also potentiated Akt activation in normal prostate epithelial cells, which were resistant to the apoptotic effects of simvastatin. Elevation of circulating cholesterol in SCID mice increased the cholesterol content and the extent of protein tyrosine phosphorylation in lipid rafts isolated from LNCaP/sHB xenograft tumors. Cholesterol elevation also promoted tumor growth, increased phosphorylation of Akt, and reduced apoptosis in the xenografts. Our results implicate membrane cholesterol in Akt signaling in both normal and malignant cells and provide evidence that PCa cells can become dependent on a cholesterol-regulated Akt pathway for cell survival.
The binding of HDL to scavenger receptor–BI (SR-BI) mediates cholesterol movement. HDL also induces multiple cellular signals, which in endothelium occur through SR-BI and converge to activate eNOS. To determine the molecular basis of a signaling event induced by HDL, we examined the proximal mechanisms in HDL activation of eNOS. In endothelial cells, HDL and methyl-β-cyclodextrin caused comparable eNOS activation, whereas cholesterol-loaded methyl-β-cyclodextrin had no effect. Phosphatidylcholine-loaded HDL caused greater stimulation than native HDL, and blocking antibody against SR-BI, which prevents cholesterol efflux, prevented eNOS activation. In a reconstitution model in COS-M6 cells, wild-type SR-BI mediated eNOS activation by both HDL and small unilamellar vesicles (SUVs), whereas the SR-BI mutant AVI, which is incapable of efflux to SUV, transmitted signal by only HDL. In addition, eNOS activation by methyl-β-cyclodextrin was SR-BI dependent. Studies of mutant and chimeric class B scavenger receptors revealed that the C-terminal cytoplasmic PDZ-interacting domain and the C-terminal transmembrane domains of SR-BI are both necessary for HDL signaling. Furthermore, we demonstrated direct binding of cholesterol to the C-terminal transmembrane domain using a photoactivated derivative of cholesterol. Thus, HDL signaling requires cholesterol binding and efflux and C-terminal domains of SR-BI, and SR-BI serves as a cholesterol sensor on the plasma membrane.
The mainstay in the management of invasive bladder cancer continues to be radical cystectomy. With regard to improvement of quality of life, however, therapies that preserve the bladder are desirable. We investigated the use of intravesical PLK-1 small interfering RNA (siRNA) against bladder cancer. Patients with bladder cancers expressing high levels of PLK-1 have a poor prognosis compared with patients with low expression. Using siRNA/cationic liposomes, the expression of endogenous PLK-1 could be suppressed in bladder cancer cells in a time- and dose-dependent manner. As a consequence, PLK-1 functions were disrupted. Inhibition of bipolar spindle formation, accumulation of cyclin B1, reduced cell proliferation, and induction of apoptosis were observed. In order to determine the efficacy of the siRNA/liposomes in vivo, we established an orthotopic mouse model using a LUC-labeled bladder cancer cell line, UM-UC-3LUC. PLK-1 siRNA was successfully transfected into the cells, reduced PLK-1 expression, and prevented the growth of bladder cancer in this mouse model. This is the first demonstration, to our knowledge, of inhibition of cancer growth in the murine bladder by intravesical siRNA/cationic liposomes. We believe intravesical siRNA instillation against bladder cancer will be useful as a therapeutic tool.
Platelet activation is a hallmark of severe preeclampsia, and platelet PGH synthase 1–derived (PGHS1-derived) thromboxane A2 (TxA2) has been implicated in its pathogenesis. However, genetic disruption of PGHS1 delays parturition. We created hypomorphic PGHS1 (PGHS1Neo/Neo) mice, in which the substantial but tissue-dependent variability in the inhibition of PGHS1-derived eicosanoids achieved by low-dose aspirin treatment is mimicked, to assess the relative impact of this strategy on hemostatic and reproductive function. Depression of platelet TxA2 by 98% in PGHS1Neo/Neo mice decreased platelet aggregation and prevented thrombosis. Similarly, depression of macrophage PGE2 by 75% was associated with selectively impaired inflammatory responses. PGF2α at 8% WT levels was sufficient to induce coordinated temporal oxytocin receptor (OTR) expression in uterus and normal ovarian luteolysis in PGHS1Neo/Neo mice at late gestation, while absence of PGHS1 expression in null mice delayed OTR induction and the programmed decrease of serum progesterone during parturition. Thus, extensive but tissue-dependent variability in PG suppression, as occurs with low-dose aspirin treatment, prevents thrombosis and impairs the inflammatory response but sustains parturition. PGHS1Neo/Neo mice provide a model of low-dose aspirin therapy that elucidates how prevention or delay of preeclampsia might be achieved without compromising reproductive function.
Systemic lupus erythematosus (SLE) T cells express high levels of cAMP response element modulator (CREM) that binds to the IL-2 promoter and represses the transcription of the IL-2 gene. This study was designed to identify pathways that lead to increased binding of CREM to the IL-2 promoter in SLE T cells. Ca2+/calmodulin–dependent kinase IV (CaMKIV) was found to be increased in the nucleus of SLE T cells and to be involved in the overexpression of CREM and its binding to the IL-2 promoter. Treatment of normal T cells with SLE serum resulted in increased expression of CREM protein, increased binding of CREM to the IL-2 promoter, and decreased IL-2 promoter activity and IL-2 production. This process was abolished when a dominant inactive form of CaMKIV was expressed in normal T cells. The effect of SLE serum resided within the IgG fraction and was specifically attributed to anti–TCR/CD3 autoantibodies. This study identifies CaMKIV as being responsible for the increased expression of CREM and the decreased production of IL-2 in SLE T cells and demonstrates that anti–TCR/CD3 antibodies present in SLE sera can account for the increased expression of CREM and the suppression of IL-2 production.
Mutations in the gene encoding hepatocyte nuclear factor-4α (HNF-4α) result in maturity-onset diabetes of the young (MODY). To determine the contribution of HNF-4α to the maintenance of glucose homeostasis by the β cell in vivo, we derived a conditional knockout of HNF-4α using the Cre-loxP system. Surprisingly, deletion of HNF-4α in β cells resulted in hyperinsulinemia in fasted and fed mice but paradoxically also in impaired glucose tolerance. Islet perifusion and calcium-imaging studies showed abnormal responses of the mutant β cells to stimulation by glucose and sulfonylureas. These phenotypes can be explained in part by a 60% reduction in expression of the potassium channel subunit Kir6.2. We demonstrate using cotransfection assays that the Kir6.2 gene is a transcriptional target of HNF-4α. Our data provide genetic evidence that HNF-4α is required in the pancreatic β cell for regulation of the pathway of insulin secretion dependent on the ATP-dependent potassium channel.
Mutations in MEF2A have been implicated in an autosomal dominant form of coronary artery disease (adCAD1). In this study we sought to determine whether severe mutations in MEF2A might also explain sporadic cases of coronary artery disease (CAD). To do this, we resequenced the coding sequence and splice sites of MEF2A in approximately 300 patients with premature CAD and failed to find causative mutations in the CAD cohort. However, we did identify the 21-bp MEF2A coding sequence deletion originally implicated in adCAD1 in 1 of 300 elderly control subjects without CAD. Further screening of approximately 1,500 additional individuals without CAD revealed 2 more subjects with the MEF2A 21-bp deletion. Genotyping of 19 family members of the 3 probands with the 21-bp deletion in MEF2A revealed that the mutation did not cosegregate with early CAD. These studies support that MEF2A mutations are not a common cause of CAD in white people and argue strongly against a role for the MEF2A 21-bp deletion in autosomal dominant CAD.
Ikaros transcription factors are essential regulators of lymphopoiesis and the development of the immune system. We now show that Ikaros is expressed in hormone-producing pituitary corticomelanotroph cells, where it binds the proopiomelanocortin promoter and regulates endogenous gene expression. Loss of Ikaros in vivo results in contraction of the pituitary corticomelanotroph population, reduced circulating adrenocorticotrophic hormone levels, and adrenal glucocorticoid insufficiency. While hemopoietic reconstitution failed to correct this hormonal deficit, the phenotype of reduced body weight and diminished survival was rescued by systemic glucocorticoid-hormone administration. Given the established immunomodulatory properties of glucocorticoid hormones, these findings reveal a novel role for Ikaros in orchestrating immune-endocrine development and function.
Effective therapies for the treatment of obesity, a key element of metabolic syndrome, are urgently needed but currently lacking. Stearoyl-CoA desaturase–1 (SCD1) is the rate-limiting enzyme catalyzing the conversion of saturated long-chain fatty acids into monounsaturated fatty acids, which are major components of triglycerides. In the current study, we tested the efficacy of pharmacological inhibition of SCD1 in controlling lipogenesis and body weight in mice. SCD1-specific antisense oligonucleotide inhibitors (ASOs) reduced SCD1 expression, reduced fatty acid synthesis and secretion, and increased fatty acid oxidization in primary mouse hepatocytes. Treatment of mice with SCD1 ASOs resulted in prevention of diet-induced obesity with concomitant reductions in SCD1 expression and the ratio of oleate to stearoyl-CoA in tissues and plasma. These changes correlated with reduced body adiposity, hepatomegaly and steatosis, and postprandial plasma insulin and glucose levels. Furthermore, SCD1 ASOs reduced de novo fatty acid synthesis, decreased expression of lipogenic genes, and increased expression of genes promoting energy expenditure in liver and adipose tissues. Thus, SCD1 inhibition represents a new target for the treatment of obesity and related metabolic disorders.
Hyperoxic acute lung injury (HALI) is characterized by a cell death response with features of apoptosis and necrosis that is inhibited by IL-11 and other interventions. We hypothesized that Bfl-1/A1, an antiapoptotic Bcl-2 protein, is a critical regulator of HALI and a mediator of IL-11–induced cytoprotection. To test this, we characterized the expression of A1 and the oxygen susceptibility of WT and IL-11 Tg(+) mice with normal and null A1 loci. In WT mice, 100% O2 caused TUNEL+ cell death, induction and activation of intrinsic and mitochondrial-death pathways, and alveolar protein leak. Bcl-2 and Bcl-xl were also induced as an apparent protective response. A1 was induced in hyperoxia, and in A1-null mice, the toxic effects of hyperoxia were exaggerated, Bcl-2 and Bcl-xl were not induced, and premature death was seen. In contrast, IL-11 stimulated A1, diminished the toxic effects of hyperoxia, stimulated Bcl-2 and Bcl-xl, and enhanced murine survival in 100% O2. In A1-null mice, IL-11–induced protection, survival advantage, and Bcl-2 and Bcl-xl induction were significantly decreased. VEGF also conferred protection via an A1-dependent mechanism. In vitro hyperoxia also stimulated A1, and A1 overexpression inhibited oxidant-induced epithelial cell apoptosis and necrosis. A1 is an important regulator of oxidant-induced lung injury, apoptosis, necrosis, and Bcl-2 and Bcl-xl gene expression and a critical mediator of IL-11– and VEGF-induced cytoprotection.
X-linked lymphoproliferative disease (XLP) is an often-fatal immunodeficiency characterized by hypogammaglobulinemia, fulminant infectious mononucleosis, and/or lymphoma. The genetic lesion in XLP, SH2D1A, encodes the adaptor protein SAP (signaling lymphocytic activation molecule–associated [SLAM-associated] protein); however, the mechanism(s) by which mutations in SH2D1A causes hypogammaglobulinemia is unknown. Our analysis of 14 XLP patients revealed normal B cell development but a marked reduction in the number of memory B cells. The few memory cells detected were IgM+, revealing deficient isotype switching in vivo. However, XLP B cells underwent proliferation and differentiation in vitro as efficiently as control B cells, which indicates that the block in differentiation in vivo is B cell extrinsic. This possibility is supported by the finding that XLP CD4+ T cells did not efficiently differentiate into IL-10+ effector cells or provide optimal B cell help in vitro. Importantly, the B cell help provided by SAP-deficient CD4+ T cells was improved by provision of exogenous IL-10 or ectopic expression of SAP, which resulted in increased IL-10 production by T cells. XLP CD4+ T cells also failed to efficiently upregulate expression of inducible costimulator (ICOS), a potent inducer of IL-10 production by CD4+ T cells. Thus, insufficient IL-10 production may contribute to hypogammaglobulinemia in XLP. This finding suggests new strategies for treating this immunodeficiency.
Osteopontin (OPN) is an extracellular matrix protein of pleiotropic properties and has been recently recognized as a potential inflammatory cytokine. In this study, we demonstrate, for the first time to our knowledge, that overexpression of OPN in synovial T cells is associated with local inflammatory milieu and that OPN acts as an important mediator in amplification and perpetuation of rheumatoid synovitis. The study revealed that mRNA expression of OPN was highly elevated in CD4+ synovial T cells derived from patients with RA, which correlated with increased OPN concentrations in synovial fluid (SF). The pattern of OPN overexpression was confined to rheumatoid synovium and correlated with coexpression of selected OPN receptors in synovial T cells, including integrins αv and β1 and CD44. RA-derived SF stimulated the expression of OPN in T cells, which was attributable to IL-10 present in SF and abrogated by anti–IL-10 antibody. Among the more than 300 autoimmune and inflammatory response genes examined, OPN selectively induced the expression of proinflammatory cytokines and chemokines known to promote migration and recruitment of inflammatory cells. Furthermore, it was evident that OPN activated transcription factor NF-κB in mononuclear cells. The study has important implications for understanding the role of OPN in rheumatoid synovitis and other inflammatory conditions.
In papillary thyroid carcinomas (PTCs), rearrangements of the RET receptor (RET/PTC) and activating mutations in the BRAF or RAS oncogenes are mutually exclusive. Here we show that the 3 proteins function along a linear oncogenic signaling cascade in which RET/PTC induces RAS-dependent BRAF activation and RAS- and BRAF-dependent ERK activation. Adoptive activation of the RET/PTC-RAS-BRAF axis induced cell proliferation and Matrigel invasion of thyroid follicular cells. Gene expression profiling revealed that the 3 oncogenes activate a common transcriptional program in thyroid cells that includes upregulation of the CXCL1 and CXCL10 chemokines, which in turn stimulate proliferation and invasion. Thus, motile and mitogenic properties are intrinsic to transformed thyroid cells and are governed by an epistatic oncogenic signaling cascade.
T cell activation levels in HIV infection are predictive of AIDS progression. We searched for the immunological correlates of protection against disease progression by studying the early stages of nonpathogenic SIV infection in African green monkeys (SIVagm). The African green monkeys (AGMs) displayed high peak viremias and a transient decline in levels of blood CD4+ and CD8+ T cells between days 5 and 17 after infection. A concomitant increase in levels of CD4+DR+, CD8+DR+, and CD8+CD28– cells was detected. After the third week, T cell activation returned to baseline levels, which suggested a protective downregulation of T cell activation. A very early (24 hours after infection) and strong induction of TGF-β1 and FoxP3 expression was detected and correlated with increases in levels of CD4+CD25+ and CD8+CD25+ T cells. This was followed by a significant increase in levels of IL-10, whereas IFN-γ gene upregulation was more transient, and levels of TNF-α and MIP-1α/β transcripts did not increase in either blood or tissues. The profiles were significantly different during primary SIV infection in macaques (SIVmac); that is, there was a delayed increase in IL-10 levels accompanied by moderate and persistent increases in TGF-β levels. Together, our data show that SIVagm infection is associated with an immediate antiinflammatory environment and suggest that TGF-β may participate in the generation of Tregs, which may prevent an aberrant chronic T cell hyperactivation.
Angiotensin II, acting through type 1 angiotensin (AT1) receptors, has potent effects that alter renal excretory mechanisms. Control of sodium excretion by the kidney has been suggested to be the critical mechanism for blood pressure regulation by the renin-angiotensin system (RAS). However, since AT1 receptors are ubiquitously expressed, precisely dissecting their physiological actions in individual tissue compartments including the kidney with conventional pharmacological or gene targeting experiments has been difficult. Here, we used a cross-transplantation strategy and AT1A receptor–deficient mice to demonstrate distinct and virtually equivalent contributions of AT1 receptor actions in the kidney and in extrarenal tissues to determining the level of blood pressure. We demonstrate that regulation of blood pressure by extrarenal AT1A receptors cannot be explained by altered aldosterone generation, which suggests that AT1 receptor actions in systemic tissues such as the vascular and/or the central nervous systems make nonredundant contributions to blood pressure regulation. We also show that interruption of the AT1 receptor–mediated short-loop feedback in the kidney is not sufficient to explain the marked stimulation of renin production induced by global AT1 receptor deficiency or by receptor blockade. Instead, the renin response seems to be primarily determined by renal baroreceptor mechanisms triggered by reduced blood pressure. Thus, the regulation of blood pressure by the RAS is mediated by AT1 receptors both within and outside the kidney.
Copyright © 2014 American Society for Clinical Investigation