Recently published - More

Abstract

Patients with a germline mutation in von Hippel-Lindau (VHL) develop renal cell cancers and hypervascular tumors of the brain, adrenal glands, and pancreas as well as erythrocytosis. These phenotypes are driven by aberrant expression of HIF2α, which induces expression of genes involved in cell proliferation, angiogenesis, and red blood cell production. Currently, there are no effective treatments available for VHL disease. Here, using an animal model of VHL, we report a marked improvement of VHL-associated phenotypes following treatment with HIF2α inhibitors. Inactivation of vhl in zebrafish led to constitutive activation of HIF2α orthologs and modeled several aspects of the human disease, including erythrocytosis, pathologic angiogenesis in the brain and retina, and aberrant kidney and liver proliferation. Treatment of vhl–/– mutant embryos with HIF2α-specific inhibitors downregulated Hif target gene expression in a dose-dependent manner, improved abnormal hematopoiesis, and substantially suppressed erythrocytosis and angiogenic sprouting. Moreover, pharmacologic inhibition of HIF2α reversed the compromised cardiac contractility of vhl–/– embryos and partially rescued early lethality. This study demonstrates that small-molecule targeting of HIF2α improves VHL-related phenotypes in a vertebrate animal model and supports further exploration of this strategy for treating VHL disease.

Authors

Ana Martins Metelo ... Randall T. Peterson, Othon Iliopoulos

×

Abstract

The polycomb protein MEL-18 has been proposed as a tumor suppressor in breast cancer; however, its functional relevance to the hormonal regulation of breast cancer remains unknown. Here, we demonstrated that MEL-18 loss contributes to the hormone-independent phenotype of breast cancer by modulating hormone receptor expression. In multiple breast cancer cohorts, MEL-18 was markedly downregulated in triple-negative breast cancer (TNBC). MEL-18 expression positively correlated with the expression of luminal markers, including estrogen receptor–α (ER-α, encoded by ESR1). MEL-18 loss was also associated with poor response to antihormonal therapy in ER-α–positive breast cancer. Furthermore, whereas MEL-18 loss in luminal breast cancer cells resulted in the downregulation of expression and activity of ER-α and the progesterone receptor (PR), MEL-18 overexpression restored ER-α expression in TNBC. Consistently, in vivo xenograft experiments demonstrated that MEL-18 loss induces estrogen-independent growth and tamoxifen resistance in luminal breast cancer, and that MEL-18 overexpression confers tamoxifen sensitivity in TNBC. MEL-18 suppressed SUMOylation of the ESR1 transactivators p53 and SP1, thereby driving ESR1 transcription. MEL-18 facilitated the deSUMOylation process by inhibiting BMI-1/RING1B-mediated ubiquitin-proteasomal degradation of SUMO1/sentrin-specific protease 1 (SENP1). These findings demonstrate that MEL-18 is a SUMO-dependent regulator of hormone receptors and suggest MEL-18 expression as a marker for determining the antihormonal therapy response in patients with breast cancer.

Authors

Jeong-Yeon Lee ... Young-Ha Oh, Gu Kong

×

Abstract

Familial forms of focal segmental glomerulosclerosis (FSGS) have been linked to gain-of-function mutations in the gene encoding the transient receptor potential channel C6 (TRPC6). GPCRs coupled to Gq signaling activate TRPC6, suggesting that Gq-dependent TRPC6 activation underlies glomerular diseases. Here, we developed a murine model in which a constitutively active Gq α subunit (GqQ209L, referred to herein as GqQ>L) is specifically expressed in podocytes and examined the effects of this mutation in response to puromycin aminonucleoside (PAN) nephrosis. We found that compared with control animals, animals expressing GqQ>L exhibited robust albuminuria, structural features of FSGS, and reduced numbers of glomerular podocytes. Gq activation stimulated calcineurin (CN) activity, resulting in CN-dependent upregulation of TRPC6 in murine kidneys. Deletion of TRPC6 in GqQ>L-expressing mice prevented FSGS development and inhibited both tubular damage and podocyte loss induced by PAN nephrosis. Similarly, administration of the CN inhibitor FK506 reduced proteinuria and tubular injury but had more modest effects on glomerular pathology and podocyte numbers in animals with constitutive Gq activation. Moreover, these Gq-dependent effects on podocyte injury were generalizable to diabetic kidney disease, as expression of GqQ>L promoted albuminuria, mesangial expansion, and increased glomerular basement membrane width in diabetic mice. Together, these results suggest that targeting Gq/TRPC6 signaling may have therapeutic benefits for the treatment of glomerular diseases.

Authors

Liming Wang ... Michelle P. Winn, Robert F. Spurney

×

Abstract

Multiple myeloma (MM) is an age-dependent hematological malignancy. Evaluation of immune interactions that drive MM relies on in vitro experiments that do not reflect the complex cellular stroma involved in MM pathogenesis. Here we used Vk*MYC transgenic mice, which spontaneously develop MM, and demonstrated that the immune system plays a critical role in the control of MM progression and the response to treatment. We monitored Vk*MYC mice that had been crossed with Cd226 mutant mice over a period of 3 years and found that CD226 limits spontaneous MM development. The CD226-dependent anti-myeloma immune response against transplanted Vk*MYC MM cells was mediated both by NK and CD8+ T cells through perforin and IFN-γ pathways. Moreover, CD226 expression was required for optimal antimyeloma efficacy of cyclophosphamide (CTX) and bortezomib (Btz), which are both standardly used to manage MM in patients. Activation of costimulatory receptor CD137 with mAb (4-1BB) exerted strong antimyeloma activity, while inhibition of coinhibitory receptors PD-1 and CTLA-4 had no effect. Taken together, the results of this study provide in vivo evidence that CD226 is important for MM immunosurveillance and indicate that specific immune components should be targeted for optimal MM treatment efficacy. As progressive immunosuppression associates with MM development, strategies aimed to increase immune functions may have important therapeutic implications in MM.

Authors

Camille Guillerey ... Mark J. Smyth, Ludovic Martinet

×

Abstract

Current strategies to alter disease-associated epigenetic modifications target ubiquitously expressed epigenetic regulators. This approach does not allow specific genes to be controlled in specific cell types; therefore, tools to selectively target epigenetic modifications in the desired cell type and strategies to more efficiently correct aberrant gene expression in disease are needed. Here, we have developed a method for directing DNA methylation to specific gene loci by conjugating catalytic domains of DNA methyltransferases (DNMTs) to engineered transcription activator–like effectors (TALEs). We demonstrated that these TALE-DNMTs direct DNA methylation specifically to the targeted gene locus in human cells. Further, we determined that minimizing direct nucleotide sequence repeats within the TALE moiety permits efficient lentivirus transduction, allowing easy targeting of primary cell types. Finally, we demonstrated that directed DNA methylation with a TALE-DNMT targeting the CDKN2A locus, which encodes the cyclin-dependent kinase inhibitor p16, decreased CDKN2A expression and increased replication of primary human fibroblasts, as intended. Moreover, overexpression of p16 in these cells reversed the proliferative phenotype, demonstrating the specificity of our epigenetic targeting. Together, our results demonstrate that TALE-DNMTs can selectively target specific genes and suggest that this strategy has potential application for the development of locus-specific epigenetic therapeutics.

Authors

Diana L. Bernstein, John E. Le Lay, Elena G. Ruano, Klaus H. Kaestner

×

Abstract

Adoptive transfer of tumor-reactive T cells can successfully reduce tumor burden; however, in rare cases, lethal on-target/off-tumor effects have been reported. A noninvasive method to track engineered cells with high sensitivity and resolution would allow observation of correct cell homing and/or identification of dangerous off-target locations in preclinical and clinical applications. Human deoxycytidine kinase triple mutant (hdCK3mut) is a nonimmunogenic PET reporter that was previously shown to be an effective tool to monitor whole-body hematopoiesis. Here, we engineered a construct in which hdCK3mut is coexpressed with the anti-melanoma T cell receptor F5, introduced this construct into human CD34 cells or PBMCs, and evaluated this approach in multiple immunotherapy models. Expression of hdCK3mut allowed engrafted cells to be visualized within recipient bone marrow, while accumulation of [18F]-L-FMAU in hdCK3mut-expressing T cells permitted detection of intratumoral homing. Animals that received T cells coexpressing hdCK3mut and the anti-melanoma T cell receptor had demonstrably higher signals in HLA-matched tumors compared with those in animals that received cells solely expressing hdCK3mut. Engineered T cells caused cytotoxicity in HLA/antigen-matched tumors and induced IFN-γ production and activation. Moreover, hdCK3mut permitted simultaneous monitoring of engraftment and tumor infiltration, without affecting T cell function. Our findings suggest that hdCK3mut reporter imaging can be applied in clinical immunotherapies for whole-body detection of engineered cell locations.

Authors

Melissa N. McCracken ... Jerome A. Zack, Owen N. Witte

×

Abstract

Activation of mTOR-dependent pathways regulates the specification and differentiation of CD4+ T effector cell subsets. Herein, we show that mTOR complex 1 (mTORC1) and mTORC2 have distinct roles in the generation of CD8+ T cell effector and memory populations. Evaluation of mice with a T cell–specific deletion of the gene encoding the negative regulator of mTORC1, tuberous sclerosis complex 2 (TSC2), resulted in the generation of highly glycolytic and potent effector CD8+ T cells; however, due to constitutive mTORC1 activation, these cells retained a terminally differentiated effector phenotype and were incapable of transitioning into a memory state. In contrast, CD8+ T cells deficient in mTORC1 activity due to loss of RAS homolog enriched in brain (RHEB) failed to differentiate into effector cells but retained memory characteristics, such as surface marker expression, a lower metabolic rate, and increased longevity. However, these RHEB-deficient memory-like T cells failed to generate recall responses as the result of metabolic defects. While mTORC1 influenced CD8+ T cell effector responses, mTORC2 activity regulated CD8+ T cell memory. mTORC2 inhibition resulted in metabolic reprogramming, which enhanced the generation of CD8+ memory cells. Overall, these results define specific roles for mTORC1 and mTORC2 that link metabolism and CD8+ T cell effector and memory generation and suggest that these functions have the potential to be targeted for enhancing vaccine efficacy and antitumor immunity.

Authors

Kristen N. Pollizzi ... Greg M. Delgoffe, Jonathan D. Powell

×

Abstract

Tumor cells can engage in a process called collective invasion, in which cohesive groups of cells invade through interstitial tissue. Here, we identified an epigenetically distinct subpopulation of breast tumor cells that have an enhanced capacity to collectively invade. Analysis of spheroid invasion in an organotypic culture system revealed that these “trailblazer” cells are capable of initiating collective invasion and promote non-trailblazer cell invasion, indicating a commensal relationship among subpopulations within heterogenous tumors. Canonical mesenchymal markers were not sufficient to distinguish trailblazer cells from non-trailblazer cells, suggesting that defining the molecular underpinnings of the trailblazer phenotype could reveal collective invasion-specific mechanisms. Functional analysis determined that DOCK10, ITGA11, DAB2, PDFGRA, VASN, PPAP2B, and LPAR1 are highly expressed in trailblazer cells and required to initiate collective invasion, with DOCK10 essential for metastasis. In patients with triple-negative breast cancer, expression of these 7 genes correlated with poor outcome. Together, our results indicate that spontaneous conversion of the epigenetic state in a subpopulation of cells can promote a transition from in situ to invasive growth through induction of a cooperative form of collective invasion and suggest that therapeutic inhibition of trailblazer cell invasion may help prevent metastasis.

Authors

Jill M. Westcott ... Yang Xie, Gray W. Pearson

×

Abstract

Microglia contribute to development, homeostasis, and immunity of the CNS. Like other tissue-resident macrophage populations, microglia express the surface receptor triggering receptor expressed on myeloid cells 2 (TREM2), which binds polyanions, such as dextran sulphate and bacterial LPS, and activates downstream signaling cascades through the adapter DAP12. Individuals homozygous for inactivating mutations in TREM2 exhibit demyelination of subcortical white matter and a lethal early onset dementia known as Nasu-Hakola disease. How TREM2 deficiency mediates demyelination and disease is unknown. Here, we addressed the basis for this genetic association using Trem2–/– mice. In WT mice, microglia expanded in the corpus callosum with age, whereas aged Trem2–/– mice had fewer microglia with an abnormal morphology. In the cuprizone model of oligodendrocyte degeneration and demyelination, Trem2–/– microglia failed to amplify transcripts indicative of activation, phagocytosis, and lipid catabolism in response to myelin damage. As a result, Trem2–/– mice exhibited impaired myelin debris clearance, axonal dystrophy, oligodendrocyte reduction, and persistent demyelination after prolonged cuprizone treatment. Moreover, myelin-associated lipids robustly triggered TREM2 signaling in vitro, suggesting that TREM2 may directly sense lipid components exposed during myelin damage. We conclude that TREM2 is required for promoting microglial expansion during aging and microglial response to insults of the white matter.

Authors

Pietro Luigi Poliani ... Susan Gilfillan, Marco Colonna

×

Abstract

BACKGROUND. The identification and treatment of individuals with tuberculosis (TB) is a global public health priority. Accurate diagnosis of pulmonary active TB (ATB) disease remains challenging and relies on extensive medical evaluation and detection of Mycobacterium tuberculosis (Mtb) in the patient’s sputum. Further, the response to treatment is monitored by sputum culture conversion, which takes several weeks for results. Here, we sought to identify blood-based host biomarkers associated with ATB and hypothesized that immune activation markers on Mtb-specific CD4+ T cells would be associated with Mtb load in vivo and could thus provide a gauge of Mtb infection.

METHODS. Using polychromatic flow cytometry, we evaluated the expression of immune activation markers on Mtb-specific CD4+ T cells from individuals with asymptomatic latent Mtb infection (LTBI) and ATB as well as from ATB patients undergoing anti-TB treatment.

RESULTS. Frequencies of Mtb-specific IFN-γ+CD4+ T cells that expressed immune activation markers CD38 and HLA-DR as well as intracellular proliferation marker Ki-67 were substantially higher in subjects with ATB compared with those with LTBI. These markers accurately classified ATB and LTBI status, with cutoff values of 18%, 60%, and 5% for CD38+IFN-γ+, HLA-DR+IFN-γ+, and Ki-67+IFN-γ+, respectively, with 100% specificity and greater than 96% sensitivity. These markers also distinguished individuals with untreated ATB from those who had successfully completed anti-TB treatment and correlated with decreasing mycobacterial loads during treatment.

CONCLUSION. We have identified host blood-based biomarkers on Mtb-specific CD4+ T cells that discriminate between ATB and LTBI and provide a set of tools for monitoring treatment response and cure.

TRIAL REGISTRATION. Registration is not required for observational studies.

FUNDING. This study was funded by Emory University, the NIH, and the Yerkes National Primate Center.

Authors

Toidi Adekambi ... Susan M. Ray, Jyothi Rengarajan

×

Abstract

Rapidly cycling fetal and neonatal hematopoietic stem cells (HSCs) generate a pool of quiescent adult HSCs after establishing hematopoiesis in the bone marrow. We report an essential role for the trithorax group gene absent, small, or homeotic 1-like (Ash1l) at this developmental transition. Emergence and expansion of Ash1l-deficient fetal/neonatal HSCs were preserved; however, in young adult animals, HSCs were profoundly depleted. Ash1l-deficient adult HSCs had markedly decreased quiescence and reduced cyclin-dependent kinase inhibitor 1b/c (Cdkn1b/1c) expression and failed to establish long-term trilineage bone marrow hematopoiesis after transplantation to irradiated recipients. Wild-type HSCs could efficiently engraft when transferred to unirradiated, Ash1l-deficient recipients, indicating increased availability of functional HSC niches in these mice. Ash1l deficiency also decreased expression of multiple Hox genes in hematopoietic progenitors. Ash1l cooperated functionally with mixed-lineage leukemia 1 (Mll1), as combined loss of Ash1l and Mll1, but not isolated Ash1l or Mll1 deficiency, induced overt hematopoietic failure. Our results uncover a trithorax group gene network that controls quiescence, niche occupancy, and self-renewal potential in adult HSCs.

Authors

Morgan Jones ... Sally A. Camper, Ivan Maillard

×

Abstract

Osteosarcoma is a common malignant bone tumor with a propensity to metastasize to the lungs. Epigenetic abnormalities have been demonstrated to underlie osteosarcoma development; however, the epigenetic mechanisms that are involved in metastasis are not yet clear. Here, we analyzed 2 syngeneic primary human osteosarcoma cell lines that exhibit disparate metastatic potential for differences in epigenetic modifications and expression. Using methylated DNA immunoprecipitation (MeDIP) and microarray expression analysis to screen for metastasis-associated genes, we identified Iroquois homeobox 1 (IRX1). In both human osteosarcoma cell lines and clinical osteosarcoma tissues, IRX1 overexpression was strongly associated with hypomethylation of its own promoter. Furthermore, experimental modulation of IRX1 in osteosarcoma cell lines profoundly altered metastatic activity, including migration, invasion, and resistance to anoikis in vitro, and influenced lung metastasis in murine models. These prometastatic effects of IRX1 were mediated by upregulation of CXCL14/NF-κB signaling. In serum from osteosarcoma patients, the presence of IRX1 hypomethylation in circulating tumor DNA reduced lung metastasis–free survival. Together, these results identify IRX1 as a prometastatic gene, implicate IRX1 hypomethylation as a potential molecular marker for lung metastasis, and suggest that epigenetic reversion of IRX1 activation may be beneficial for controlling osteosarcoma metastasis.

Authors

Jinchang Lu ... Jingnan Shen, Jin Wang

×

Abstract

DCs are able to undergo rapid maturation, which subsequently allows them to initiate and orchestrate T cell–driven immune responses. DC maturation must be tightly controlled in order to avoid random T cell activation and development of autoimmunity. Here, we determined that 12/15-lipoxygenase–meditated (12/15-LO–mediated) enzymatic lipid oxidation regulates DC activation and fine-tunes consecutive T cell responses. Specifically, 12/15-LO activity determined the DC activation threshold via generation of phospholipid oxidation products that induced an antioxidative response dependent on the transcription factor NRF2. Deletion of the 12/15-LO–encoding gene or pharmacologic inhibition of 12/15-LO in murine or human DCs accelerated maturation and shifted the cytokine profile, thereby favoring the differentiation of Th17 cells. Exposure of 12/15-LO–deficient DCs to 12/15-LO–derived oxidized phospholipids attenuated both DC activation and the development of Th17 cells. Analysis of lymphatic tissues from 12/15-LO–deficient mice confirmed enhanced maturation of DCs as well as an increased differentiation of Th17 cells. Moreover, experimental autoimmune encephalomyelitis in mice lacking 12/15-LO resulted in an exacerbated Th17-driven autoimmune disease. Together, our data reveal that 12/15-LO controls maturation of DCs and implicate enzymatic lipid oxidation in shaping the adaptive immune response.

Authors

Tobias Rothe ... Elisabeth Zinser, Gerhard Krönke

×

Abstract

Thiazide diuretics are used to treat hypertension; however, compensatory processes in the kidney can limit antihypertensive responses to this class of drugs. Here, we evaluated compensatory pathways in SPAK kinase–deficient mice, which are unable to activate the thiazide-sensitive sodium chloride cotransporter NCC (encoded by Slc12a3). Global transcriptional profiling, combined with biochemical, cell biological, and physiological phenotyping, identified the gene expression signature of the response and revealed how it establishes an adaptive physiology. Salt reabsorption pathways were created by the coordinate induction of a multigene transport system, involving solute carriers (encoded by Slc26a4, Slc4a8, and Slc4a9), carbonic anhydrase isoforms, and V-type H+-ATPase subunits in pendrin-positive intercalated cells (PP-ICs) and ENaC subunits in principal cells (PCs). A distal nephron remodeling process and induction of jagged 1/NOTCH signaling, which expands the cortical connecting tubule with PCs and replaces acid-secreting α-ICs with PP-ICs, were partly responsible for the compensation. Salt reabsorption was also activated by induction of an α-ketoglutarate (α-KG) paracrine signaling system. Coordinate regulation of a multigene α-KG synthesis and transport pathway resulted in α-KG secretion into pro-urine, as the α-KG–activated GPCR (Oxgr1) increased on the PP-IC apical surface, allowing paracrine delivery of α-KG to stimulate salt transport. Identification of the integrated compensatory NaCl reabsorption mechanisms provides insight into thiazide diuretic efficacy.

Authors

P. Richard Grimm ... James B. Wade, Paul A. Welling

×

Abstract

Myelodysplastic syndromes and chronic myelomonocytic leukemia (CMML) are characterized by mutations in genes encoding epigenetic modifiers and aberrant DNA methylation. DNA methyltransferase inhibitors (DMTis) are used to treat these disorders, but response is highly variable, with few means to predict which patients will benefit. Here, we examined baseline differences in mutations, DNA methylation, and gene expression in 40 CMML patients who were responsive or resistant to decitabine (DAC) in order to develop a molecular means of predicting response at diagnosis. While somatic mutations did not differentiate responders from nonresponders, we identified 167 differentially methylated regions (DMRs) of DNA at baseline that distinguished responders from nonresponders using next-generation sequencing. These DMRs were primarily localized to nonpromoter regions and overlapped with distal regulatory enhancers. Using the methylation profiles, we developed an epigenetic classifier that accurately predicted DAC response at the time of diagnosis. Transcriptional analysis revealed differences in gene expression at diagnosis between responders and nonresponders. In responders, the upregulated genes included those that are associated with the cell cycle, potentially contributing to effective DAC incorporation. Treatment with CXCL4 and CXCL7, which were overexpressed in nonresponders, blocked DAC effects in isolated normal CD34+ and primary CMML cells, suggesting that their upregulation contributes to primary DAC resistance.

Authors

Kristen Meldi ... Valeria Santini, Maria E. Figueroa

×

Abstract

The HSP40 cochaperone SEC63 is associated with the SEC61 translocon complex in the ER. Mutations in the gene encoding SEC63 cause polycystic liver disease in humans; however, it is not clear how altered SEC63 influences disease manifestations. In mice, loss of SEC63 induces cyst formation both in liver and kidney as the result of reduced polycystin-1 (PC1). Here we report that inactivation of SEC63 induces an unfolded protein response (UPR) pathway that is protective against cyst formation. Specifically, using murine genetic models, we determined that SEC63 deficiency selectively activates the IRE1α-XBP1 branch of UPR and that SEC63 exists in a complex with PC1. Concomitant inactivation of both SEC63 and XBP1 exacerbated the polycystic kidney phenotype in mice by markedly suppressing cleavage at the G protein–coupled receptor proteolysis site (GPS) in PC1. Enforced expression of spliced XBP1 (XBP1s) enhanced GPS cleavage of PC1 in SEC63-deficient cells, and XBP1 overexpression in vivo ameliorated cystic disease in a murine model with reduced PC1 function that is unrelated to SEC63 inactivation. Collectively, the findings show that SEC63 function regulates IRE1α/XBP1 activation, SEC63 and XBP1 are required for GPS cleavage and maturation of PC1, and activation of XBP1 can protect against polycystic disease in the setting of impaired biogenesis of PC1.

Authors

Sorin V. Fedeles ... Stefan Somlo, Ann-Hwee Lee

×

Abstract

Dyskeratosis congenita (DC) and related syndromes are inherited, life-threatening bone marrow (BM) failure disorders, and approximately 40% of cases are currently uncharacterized at the genetic level. Here, using whole exome sequencing (WES), we have identified biallelic mutations in the gene encoding poly(A)-specific ribonuclease (PARN) in 3 families with individuals exhibiting severe DC. PARN is an extensively characterized exonuclease with deadenylation activity that controls mRNA stability in part and therefore regulates expression of a large number of genes. The DC-associated mutations identified affect key domains within the protein, and evaluation of patient cells revealed reduced deadenylation activity. This deadenylation deficiency caused an early DNA damage response in terms of nuclear p53 regulation, cell-cycle arrest, and reduced cell viability upon UV treatment. Individuals with biallelic PARN mutations and PARN-depleted cells exhibited reduced RNA levels for several key genes that are associated with telomere biology, specifically TERC, DKC1, RTEL1, and TERF1. Moreover, PARN-deficient cells also possessed critically short telomeres. Collectively, these results identify a role for PARN in telomere maintenance and demonstrate that it is a disease-causing gene in a subset of patients with severe DC.

Authors

Hemanth Tummala ... Thomas Vulliamy, Inderjeet Dokal

×

Abstract

Constitutively active MYC and reactivated telomerase often coexist in cancers. While reactivation of telomerase is thought to be essential for replicative immortality, MYC, in conjunction with cofactors, confers several growth advantages to cancer cells. It is known that the reactivation of TERT, the catalytic subunit of telomerase, is limiting for reconstituting telomerase activity in tumors. However, while reactivation of TERT has been functionally linked to the acquisition of several “hallmarks of cancer” in tumors, the molecular mechanisms by which this occurs and whether these mechanisms are distinct from the role of telomerase on telomeres is not clear. Here, we demonstrated that first-generation TERT-null mice, unlike Terc-null mice, show delayed onset of MYC-induced lymphomagenesis. We further determined that TERT is a regulator of MYC stability in cancer. TERT stabilized MYC levels on chromatin, contributing to either activation or repression of its target genes. TERT regulated MYC ubiquitination and proteasomal degradation, and this effect of TERT was independent of its reverse transcriptase activity and role in telomere elongation. Based on these data, we conclude that reactivation of TERT, a direct transcriptional MYC target in tumors, provides a feed-forward mechanism to potentiate MYC-dependent oncogenesis.

Authors

Cheryl M. Koh ... Ernesto Guccione, Vinay Tergaonkar

×

Abstract

The type 2 ryanodine receptor (RyR2) is a Ca2+ release channel on the endoplasmic reticulum (ER) of several types of cells, including cardiomyocytes and pancreatic β cells. In cardiomyocytes, RyR2-dependent Ca2+ release is critical for excitation-contraction coupling; however, a functional role for RyR2 in β cell insulin secretion and diabetes mellitus remains controversial. Here, we took advantage of rare RyR2 mutations that were identified in patients with a genetic form of exercise-induced sudden death (catecholaminergic polymorphic ventricular tachycardia [CPVT]). As these mutations result in a “leaky” RyR2 channel, we exploited them to assess RyR2 channel function in β cell dynamics. We discovered that CPVT patients with mutant leaky RyR2 present with glucose intolerance, which was heretofore unappreciated. In mice, transgenic expression of CPVT-associated RyR2 resulted in impaired glucose homeostasis, and an in-depth evaluation of pancreatic islets and β cells from these animals revealed intracellular Ca2+ leak via oxidized and nitrosylated RyR2 channels, activated ER stress response, mitochondrial dysfunction, and decreased fuel-stimulated insulin release. Additionally, we verified the effects of the pharmacological inhibition of intracellular Ca2+ leak in CPVT-associated RyR2-expressing mice, in human islets from diabetic patients, and in an established murine model of type 2 diabetes mellitus. Taken together, our data indicate that RyR2 channels play a crucial role in the regulation of insulin secretion and glucose homeostasis.

Authors

Gaetano Santulli ... Alain Lacampagne, Andrew R. Marks

×

Abstract

Parkin and the glial cell line–derived neurotrophic factor (GDNF) receptor RET have both been independently linked to the dopaminergic neuron degeneration that underlies Parkinson’s disease (PD). In the present study, we demonstrate that there is genetic crosstalk between parkin and the receptor tyrosine kinase RET in two different mouse models of PD. Mice lacking both parkin and RET exhibited accelerated dopaminergic cell and axonal loss compared with parkin-deficient animals, which showed none, and RET-deficient mice, in which we found moderate degeneration. Transgenic expression of parkin protected the dopaminergic systems of aged RET-deficient mice. Downregulation of either parkin or RET in neuronal cells impaired mitochondrial function and morphology. Parkin expression restored mitochondrial function in GDNF/RET-deficient cells, while GDNF stimulation rescued mitochondrial defects in parkin-deficient cells. In both cases, improved mitochondrial function was the result of activation of the prosurvival NF-κB pathway, which was mediated by RET through the phosphoinositide-3-kinase (PI3K) pathway. Taken together, these observations indicate that parkin and the RET signaling cascade converge to control mitochondrial integrity and thereby properly maintain substantia nigra pars compacta dopaminergic neurons and their innervation in the striatum. The demonstration of crosstalk between parkin and RET highlights the interplay in the protein network that is altered in PD and suggests potential therapeutic targets and strategies to treat PD.

Authors

Durga Praveen Meka ... Konstanze F. Winklhofer, Edgar R. Kramer

×

Abstract

Liver cholestatic diseases, which stem from diverse etiologies, result in liver toxicity and fibrosis and may progress to cirrhosis and liver failure. We show that CCN1 (also known as CYR61), a matricellular protein that dampens and resolves liver fibrosis, also mediates cholangiocyte proliferation and ductular reaction, which are repair responses to cholestatic injury. In cholangiocytes, CCN1 activated NF-κB through integrin αvβ5vβ3, leading to Jag1 expression, JAG1/NOTCH signaling, and cholangiocyte proliferation. CCN1 also induced Jag1 expression in hepatic stellate cells, whereupon they interacted with hepatic progenitor cells to promote their differentiation into cholangiocytes. Administration of CCN1 protein or soluble JAG1 induced cholangiocyte proliferation in mice, which was blocked by inhibitors of NF-κB or NOTCH signaling. Knock-in mice expressing a CCN1 mutant that is unable to bind αvβ5vβ3 were impaired in ductular reaction, leading to massive hepatic necrosis and mortality after bile duct ligation (BDL), whereas treatment of these mice with soluble JAG1 rescued ductular reaction and reduced hepatic necrosis and mortality. Blockade of integrin αvβ5vβ3, NF-κB, or NOTCH signaling in WT mice also resulted in defective ductular reaction after BDL. These findings demonstrate that CCN1 induces cholangiocyte proliferation and ductular reaction and identify CCN1/αvβ5/NF-κB/JAG1 as a critical axis for biliary injury repair.

Authors

Ki-Hyun Kim, Chih-Chiun Chen, Gianfranco Alpini, Lester F. Lau

×

Abstract

Epithelial cells that line the conducting airways provide the initial barrier and innate immune responses to the abundant particles, microbes, and allergens that are inhaled throughout life. The transcription factors SPDEF and FOXA3 are both selectively expressed in epithelial cells lining the conducting airways, where they regulate goblet cell differentiation and mucus production. Moreover, these transcription factors are upregulated in chronic lung disorders, including asthma. Here, we show that expression of SPDEF or FOXA3 in airway epithelial cells in neonatal mice caused goblet cell differentiation, spontaneous eosinophilic inflammation, and airway hyperresponsiveness to methacholine. SPDEF expression promoted DC recruitment and activation in association with induction of Il33, Csf2, thymic stromal lymphopoietin (Tslp), and Ccl20 transcripts. Increased Il4, Il13, Ccl17, and Il25 expression was accompanied by recruitment of Th2 lymphocytes, group 2 innate lymphoid cells, and eosinophils to the lung. SPDEF was required for goblet cell differentiation and pulmonary Th2 inflammation in response to house dust mite (HDM) extract, as both were decreased in neonatal and adult Spdef–/– mice compared with control animals. Together, our results indicate that SPDEF causes goblet cell differentiation and Th2 inflammation during postnatal development and is required for goblet cell metaplasia and normal Th2 inflammatory responses to HDM aeroallergen.

Authors

Priya Rajavelu, Gang Chen, Yan Xu, Joseph A. Kitzmiller, Thomas R. Korfhagen, Jeffrey A. Whitsett

×

Abstract

The G protein–coupled estrogen receptor (GPER) mediates both the genomic and nongenomic effects of estrogen and has been implicated in breast cancer development. Here, we compared GPER expression in cancerous tissue and adjacent normal tissue in patients with invasive ductal carcinoma (IDC) of the breast and determined that GPER is highly upregulated in cancerous cells. Additionally, our studies revealed that GPER stimulation activates yes-associated protein 1 (YAP) and transcriptional coactivator with a PDZ-binding domain (TAZ), 2 homologous transcription coactivators and key effectors of the Hippo tumor suppressor pathway, via the Gαq-11, PLCβ/PKC, and Rho/ROCK signaling pathways. TAZ was required for GPER-induced gene transcription, breast cancer cell proliferation and migration, and tumor growth. Moreover, TAZ expression positively correlated with GPER expression in human IDC specimens. Together, our results suggest that the Hippo/YAP/TAZ pathway is a key downstream signaling branch of GPER and plays a critical role in breast tumorigenesis.

Authors

Xin Zhou ... Qun-Ying Lei, Kun-Liang Guan

×

Abstract

Hematopoietic stem cells (HSCs) first emerge during embryonic development within vessels such as the dorsal aorta of the aorta-gonad-mesonephros (AGM) region, suggesting that signals from the vascular microenvironment are critical for HSC development. Here, we demonstrated that AGM-derived endothelial cells (ECs) engineered to constitutively express AKT (AGM AKT-ECs) can provide an in vitro niche that recapitulates embryonic HSC specification and amplification. Specifically, nonengrafting embryonic precursors, including the VE-cadherin–expressing population that lacks hematopoietic surface markers, cocultured with AGM AKT-ECs specified into long-term, adult-engrafting HSCs, establishing that a vascular niche is sufficient to induce the endothelial-to-HSC transition in vitro. Subsequent to hematopoietic induction, coculture with AGM AKT-ECs also substantially increased the numbers of HSCs derived from VE-cadherin+CD45+ AGM hematopoietic cells, consistent with a role in supporting further HSC maturation and self-renewal. We also identified conditions that included NOTCH activation with an immobilized NOTCH ligand that were sufficient to amplify AGM-derived HSCs following their specification in the absence of AGM AKT-ECs. Together, these studies begin to define the critical niche components and resident signals required for HSC induction and self-renewal ex vivo, and thus provide insight for development of defined in vitro systems targeted toward HSC generation for therapeutic applications.

Authors

Brandon K. Hadland ... Shahin Rafii, Irwin D. Bernstein

×

Abstract

Reversal of HIV-1 latency by small molecules is a potential cure strategy. This approach will likely require effective drug combinations to achieve high levels of latency reversal. Using resting CD4+ T cells (rCD4s) from infected individuals, we developed an experimental and theoretical framework to identify effective latency-reversing agent (LRA) combinations. Utilizing ex vivo assays for intracellular HIV-1 mRNA and virion production, we compared 2-drug combinations of leading candidate LRAs and identified multiple combinations that effectively reverse latency. We showed that protein kinase C agonists in combination with bromodomain inhibitor JQ1 or histone deacetylase inhibitors robustly induce HIV-1 transcription and virus production when directly compared with maximum reactivation by T cell activation. Using the Bliss independence model to quantitate combined drug effects, we demonstrated that these combinations synergize to induce HIV-1 transcription. This robust latency reversal occurred without release of proinflammatory cytokines by rCD4s. To extend the clinical utility of our findings, we applied a mathematical model that estimates in vivo changes in plasma HIV-1 RNA from ex vivo measurements of virus production. Our study reconciles diverse findings from previous studies, establishes a quantitative experimental approach to evaluate combinatorial LRA efficacy, and presents a model to predict in vivo responses to LRAs.

Authors

Gregory M. Laird ... Janet D. Siliciano, Robert F. Siliciano

×

Abstract

T cell Ig and ITIM domain (TIGIT) is an inhibitory receptor expressed by activated T cells, Tregs, and NK cells. Here, we determined that TIGIT is upregulated on tumor antigen–specific (TA-specific) CD8+ T cells and CD8+ tumor-infiltrating lymphocytes (TILs) from patients with melanoma, and these TIGIT-expressing CD8+ T cells often coexpress the inhibitory receptor PD-1. Moreover, CD8+ TILs from patients exhibited downregulation of the costimulatory molecule CD226, which competes with TIGIT for the same ligand, supporting a TIGIT/CD226 imbalance in metastatic melanoma. TIGIT marked early T cell activation and was further upregulated by T cells upon PD-1 blockade and in dysfunctional PD-1+TIM-3+ TA-specific CD8+ T cells. PD-1+TIGIT+, PD-1TIGIT+, and PD-1+TIGIT CD8+ TILs had similar functional capacities ex vivo, suggesting that TIGIT alone, or together with PD-1, is not indicative of T cell dysfunction. However, in the presence of TIGIT ligand–expressing cells, TIGIT and PD-1 blockade additively increased proliferation, cytokine production, and degranulation of both TA-specific CD8+ T cells and CD8+ TILs. Collectively, our results show that TIGIT and PD-1 regulate the expansion and function of TA-specific CD8+ T cells and CD8+ TILs in melanoma patients and suggest that dual TIGIT and PD-1 blockade should be further explored to elicit potent antitumor CD8+ T cell responses in patients with advanced melanoma.

Authors

Joe-Marc Chauvin ... Alan J. Korman, Hassane M. Zarour

×

Abstract

There is large variation in lifespan among different species, and there is evidence that modulation of proteasome function may contribute to longevity determination. Comparative biology provides a powerful tool for identifying genes and pathways that control the rate of aging. Here, we evaluated skin-derived fibroblasts and demonstrate that among primate species, longevity correlated with an elevation in proteasomal activity as well as immunoproteasome expression at both the mRNA and protein levels. Immunoproteasome enhancement occurred with a concurrent increase in other elements involved in MHC class I antigen presentation, including β-2 microglobulin, (TAP1), and TAP2. Fibroblasts from long-lived primates also appeared more responsive to IFN-γ than cells from short-lived primate species, and this increase in IFN-γ responsiveness correlated with elevated expression of the IFN-γ receptor protein IFNGR2. Elevation of immunoproteasome and proteasome activity was also observed in the livers of long-lived Snell dwarf mice and in mice exposed to drugs that have been shown to extend lifespan, including rapamycin, 17-α-estradiol, and nordihydroguaiaretic acid. This work suggests that augmented immunoproteasome function may contribute to lifespan differences in mice and among primate species.

Authors

Andrew M. Pickering, Marcus Lehr, Richard A. Miller

×

Abstract

Angelman syndrome (AS) is a severe neurodevelopmental disorder that results from loss of function of the maternal ubiquitin protein ligase E3A (UBE3A) allele. Due to neuron-specific imprinting, the paternal UBE3A copy is silenced. Previous studies in murine models have demonstrated that strategies to activate the paternal Ube3a allele are feasible; however, a recent study showed that pharmacological Ube3a gene reactivation in adulthood failed to rescue the majority of neurocognitive phenotypes in a murine AS model. Here, we performed a systematic study to investigate the possibility that neurocognitive rescue can be achieved by reinstating Ube3a during earlier neurodevelopmental windows. We developed an AS model that allows for temporally controlled Cre-dependent induction of the maternal Ube3a allele and determined that there are distinct neurodevelopmental windows during which Ube3a restoration can rescue AS-relevant phenotypes. Motor deficits were rescued by Ube3a reinstatement in adolescent mice, whereas anxiety, repetitive behavior, and epilepsy were only rescued when Ube3a was reinstated during early development. In contrast, hippocampal synaptic plasticity could be restored at any age. Together, these findings suggest that Ube3a reinstatement early in development may be necessary to prevent or rescue most AS-associated phenotypes and should be considered in future clinical trial design.

Authors

Sara Silva-Santos ... Steven A. Kushner, Ype Elgersma

×

Abstract

BACKGROUND: Quantification of disease-associated proteins in the cerebrospinal fluid (CSF) has been critical for the study and treatment of several neurodegenerative disorders; however, mutant huntingtin protein (mHTT), the cause of Huntington’s disease (HD), is at very low levels in CSF and, to our knowledge, has never been measured previously.

METHODS: We developed an ultrasensitive single-molecule counting (SMC) mHTT immunoassay that was used to quantify mHTT levels in CSF samples from individuals bearing the HD mutation and from control individuals in 2 independent cohorts.

RESULTS: This SMC mHTT immunoassay demonstrated high specificity for mHTT, high sensitivity with a femtomolar detection threshold, and a broad dynamic range. Analysis of the CSF samples showed that mHTT was undetectable in CSF from all controls but quantifiable in nearly all mutation carriers. The mHTT concentration in CSF was approximately 3-fold higher in patients with manifest HD than in premanifest mutation carriers. Moreover, mHTT levels increased as the disease progressed and were associated with 5-year onset probability. The mHTT concentration independently predicted cognitive and motor dysfunction. Furthermore, the level of mHTT was associated with the concentrations of tau and neurofilament light chain in the CSF, suggesting a neuronal origin for the detected mHTT.

CONCLUSIONS: We have demonstrated that mHTT can be quantified in CSF from HD patients using the described SMC mHTT immunoassay. Moreover, the level of mHTT detected is associated with proximity to disease onset and diminished cognitive and motor function. The ability to quantify CSF mHTT will facilitate the study of HD, and mHTT quantification could potentially serve as a biomarker for the development and testing of experimental mHTT-lowering therapies for HD.

TRIAL REGISTRATION: Not applicable.

FUNDING: CHDI Foundation Inc.; Medical Research Council (MRC) UK; National Institutes for Health Research (NIHR); Rosetrees Trust; Swedish Research Council; and Knut and Alice Wallenberg Foundation.

Authors

Edward J. Wild ... Douglas Macdonald, Andreas Weiss

×

Abstract

BACKGROUND. There is frequent uncertainty in the identification of specific etiologies of chronic kidney disease (CKD) in children. Recent studies indicate that chromosomal microarrays can identify rare genomic imbalances that can clarify the etiology of neurodevelopmental and cardiac disorders in children; however, the contribution of unsuspected genomic imbalance to the incidence of pediatric CKD is unknown.

METHODS. We performed chromosomal microarrays to detect genomic imbalances in children enrolled in the Chronic Kidney Disease in Children (CKiD) prospective cohort study, a longitudinal prospective multiethnic observational study of North American children with mild to moderate CKD. Patients with clinically detectable syndromic disease were excluded from evaluation. We compared 419 unrelated children enrolled in CKiD to multiethnic cohorts of 21,575 children and adults that had undergone microarray genotyping for studies unrelated to CKD.

RESULTS. We identified diagnostic copy number disorders in 31 children with CKD (7.4% of the cohort). We detected 10 known pathogenic genomic disorders, including the 17q12 deletion HNF1 homeobox B (HNF1B) and triple X syndromes in 19 of 419 unrelated CKiD cases as compared with 98 of 21,575 control individuals (OR 10.8, P = 6.1 × 10–20). In an additional 12 CKiD cases, we identified 12 likely pathogenic genomic imbalances that would be considered reportable in a clinical setting. These genomic imbalances were evenly distributed among patients diagnosed with congenital and noncongenital forms of CKD. In the vast majority of these cases, the genomic lesion was unsuspected based on the clinical assessment and either reclassified the disease or provided information that might have triggered additional clinical care, such as evaluation for metabolic or neuropsychiatric disease.

CONCLUSION. A substantial proportion of children with CKD have an unsuspected genomic imbalance, suggesting genomic disorders as a risk factor for common forms of pediatric nephropathy. Detection of pathogenic imbalances has practical implications for personalized diagnosis and health monitoring in this population.

TRIAL REGISTRATION. ClinicalTrials.gov NCT00327860.

FUNDING. This work was supported by the NIH, the National Institutes of Diabetes and Digestive and Kidney Diseases (NIDDK), the National Institute of Child Health and Human Development, and the National Heart, Lung, and Blood Institute.

Authors

Miguel Verbitsky ... Craig S. Wong, Ali G. Gharavi

×

Abstract

Diuretics are commonly used to treat hypertension and extracellular fluid volume expansion. However, the development of compensatory responses in the kidney limits the benefit of this class of drugs. In this issue of the JCI, Grimm and colleagues use a systems biology approach in mice lacking the kinase SPAK and unravel a complex mechanism that explains thiazide diuretic resistance. The overall process involves interactions among six different cell types in the kidney.

Authors

Mark A. Knepper

×

Abstract

Dyskeratosis congenita (DC) is an inherited BM failure disorder that is associated with mutations in genes involved with telomere function and maintenance; however, the genetic cause of many instances of DC remains uncharacterized. In this issue of the JCI, Tummala and colleagues identify mutations in the gene encoding the poly(A)-specific ribonuclease (PARN) in individuals with a severe form of DC in three different families. PARN deficiency resulted in decreased expression of genes required for telomere maintenance and an aberrant DNA damage response, including increased levels of p53. Together, the results of this study support PARN as a DC-associated gene and suggest a potential link between p53 and telomere shortening.

Authors

Philip J. Mason, Monica Bessler

×

Abstract

In the early 1980s, we analyzed the metabolic profile of 930 men and women and concluded that an abdominal distribution of fat for a given BMI is associated with increased insulin resistance and risk of developing type 2 diabetes and cardiovascular disease. The correlation between abdominal fat and metabolic dysfunction has since been validated in many studies, and waist circumference is now a criterion for the diagnosis of metabolic syndrome. Several mechanisms for this relationship have been postulated; however, we now know that visceral fat is only one of many ectopic fat depots used when the subcutaneous adipose tissue cannot accommodate excess fat because of its limited expandability.

Authors

Ulf Smith

×

Abstract

In children, chronic kidney disease (CKD) that results from structural abnormalities and glomerular injury is readily diagnosed; however, most cases of pediatric CKD are of unknown etiology. In this issue of the JCI, Verbitsky and colleagues used chromosomal microarrays to evaluate genomic variation in children with CKD. Compared with control individuals, a substantial proportion of children with idiopathic CKD had clearly identifiable genomic imbalances. Moreover, in some cases, detailed analysis of these imbalances identified pathogenic alterations that were unsuspected based on clinical presentation. The results of this study support genome-wide evaluation for pediatric cases of CKD; however, more work will need to be done before such an approach is widely available in the clinic.

Authors

Martin R. Pollak

×




Advertisement

May 2015


125-5-cover

May 2015 Issue

On the cover:
Visualizing tumor-infiltrating T cells

The cover image is a modified 3D reconstruction of an [18F]-L-FMAU PET/CT scan to detect hematopoietic cells labeled with a reporter gene, showing tumor-infiltrating cells within the left flank and engraftment of cells within the BM. On page 1815, McCracken et al. describe a noninvasive technique for visualizing engineered immune cells in multiple mouse models of adoptive cellular immunotherapy.

×
Jci_impact_2015_05

May 2015 Impact

JCI Impact is a digest of the research, reviews, and other features published in each month's issue of the Journal of Clinical Investigation.

×

Review Series - More

Enteric Nervous System

Series edited by Rodger Liddle

The enteric nervous system (ENS) encompasses extrinsic and intrinsic neurons, glia, and sensory epithelial cells that are embedded throughout the gastrointestinal tract. The circuits formed by these cells are responsible for interpreting sensory information in the gut lumen in order to regulate gut motility, secretion, food intake, and immune function. The ENS communicates with the CNS in a bidirectional manner, allowing stimuli in the gut to influence mood, food intake, and other behaviors. Reviews in this series examine the mechanisms by which the ENS develops from neural crest cells, chemosensory mechanisms that allow for the detection of and response to fats and other nutrients within the gut lumen, the role of the enteric glia, regulation of ENS function by the immune system and inflammation, and the impact of surgery and the gut microbiota on ENS communication with the brain.

×