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Abstract

Airway hyperresponsiveness (AHR) affects 55%–77% of children with sickle cell disease (SCD) and occurs even in the absence of asthma. While asthma increases SCD morbidity and mortality, the mechanisms underlying the high AHR prevalence in a hemoglobinopathy remain unknown. We hypothesized that placenta growth factor (PlGF), an erythroblast-secreted factor that is elevated in SCD, mediates AHR. In allergen-exposed mice, loss of Plgf dampened AHR, reduced inflammation and eosinophilia, and decreased expression of the Th2 cytokine IL-13 and the leukotriene-synthesizing enzymes 5-lipoxygenase and leukotriene-C4-synthase. Plgf–/– mice treated with leukotrienes phenocopied the WT response to allergen exposure; conversely, anti-PlGF Ab administration in WT animals blunted the AHR. Notably, Th2-mediated STAT6 activation further increased PlGF expression from lung epithelium, eosinophils, and macrophages, creating a PlGF/leukotriene/Th2-response positive feedback loop. Similarly, we found that the Th2 response in asthma patients is associated with increased expression of PlGF and its downstream genes in respiratory epithelial cells. In an SCD mouse model, we observed increased AHR and higher leukotriene levels that were abrogated by anti-PlGF Ab or the 5-lipoxygenase inhibitor zileuton. Overall, our findings indicate that PlGF exacerbates AHR and uniquely links the leukotriene and Th2 pathways in asthma. These data also suggest that zileuton and anti-PlGF Ab could be promising therapies to reduce pulmonary morbidity in SCD.

Authors

Marthe-Sandrine Eiymo Mwa Mpollo, Eric B. Brandt, Shiva Kumar Shanmukhappa, Paritha I. Arumugam, Swati Tiwari, Anastacia Loberg, Devin Pillis, Tilat Rizvi, Mark Lindsey, Bart Jonck, Peter Carmeliet, Vijay K. Kalra, Timothy D. Le Cras, Nancy Ratner, Marsha Wills-Karp, Gurjit K. Khurana Hershey, Punam Malik

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Abstract

Histone demethylase upregulation has been observed in human cancers, yet it is unknown whether this is a bystander event or a driver of tumorigenesis. We found that overexpression of lysine-specific demethylase 4A (KDM4A, also known as JMJD2A) was positively correlated with Gleason score and metastasis in human prostate tumors. Overexpression of JMJD2A resulted in the development of prostatic intraepithelial neoplasia in mice, demonstrating that JMJD2A can initiate prostate cancer development. Moreover, combined overexpression of JMJD2A and the ETS transcription factor ETV1, a JMJD2A-binding protein, resulted in prostate carcinoma formation in mice haplodeficient for the phosphatase and tensin homolog (Pten) tumor-suppressor gene. Additionally, JMJD2A cooperated with ETV1 to increase expression of yes associated protein 1 (YAP1), a Hippo pathway component that itself was associated with prostate tumor aggressiveness. ETV1 facilitated the recruitment of JMJD2A to the YAP1 promoter, leading to changes in histone lysine methylation in a human prostate cancer cell line. Further, YAP1 expression largely rescued the growth inhibitory effects of JMJD2A depletion in prostate cancer cells, indicating that YAP1 is a downstream effector of JMJD2A. Taken together, these data reveal a JMJD2A/ETV1/YAP1 axis that promotes prostate cancer initiation and that may be a suitable target for therapeutic inhibition.

Authors

Tae-Dong Kim, Fang Jin, Sook Shin, Sangphil Oh, Stan A. Lightfoot, Joseph P. Grande, Aaron J. Johnson, Jan M. van Deursen, Jonathan D. Wren, Ralf Janknecht

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Abstract

Analogous behavioral assays are needed across animal models and human patients to improve translational research. Here, we examined the extent to which performance in the Morris water maze — the most frequently used behavioral assay of spatial learning and memory in rodents — translates to humans. We designed a virtual version of the assay for human subjects that includes the visible-target training, hidden-target learning, and probe trials that are typically administered in the mouse version. We compared transgenic mice that express human amyloid precursor protein (hAPP) and patients with mild cognitive impairment due to Alzheimer’s disease (MCI-AD) to evaluate the sensitivity of performance measures in detecting deficits. Patients performed normally during visible-target training, while hAPP mice showed procedural learning deficits. In hidden-target learning and probe trials, hAPP mice and MCI-AD patients showed similar deficits in learning and remembering the target location. In addition, we have provided recommendations for selecting performance measures and sample sizes to make these assays sensitive to learning and memory deficits in humans with MCI-AD and in mouse models. Together, our results demonstrate that with careful study design and analysis, the Morris maze is a sensitive assay for detecting AD-relevant impairments across species.

Authors

Katherine L. Possin, Pascal E. Sanchez, Clifford Anderson-Bergman, Roland Fernandez, Geoffrey A. Kerchner, Erica T. Johnson, Allyson Davis, Iris Lo, Nicholas T. Bott, Thomas Kiely, Michelle C. Fenesy, Bruce L. Miller, Joel H. Kramer, Steven Finkbeiner

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Abstract

The maintenance of skeletal muscle mass is critical for sustaining health; however, the mechanisms responsible for muscle loss with aging and chronic diseases, such as diabetes and obesity, are poorly understood. We found that expression of a member of the AMPK-related kinase family, the SNF1-AMPK-related kinase (SNARK, also known as NUAK2), increased with muscle cell differentiation. SNARK expression increased in skeletal muscles from young mice exposed to metabolic stress and in muscles from healthy older human subjects. The regulation of SNARK expression in muscle with differentiation and physiological stress suggests that SNARK may function in the maintenance of muscle mass. Consistent with this hypothesis, decreased endogenous SNARK expression (using siRNA) in cultured muscle cells resulted in increased apoptosis and decreased cell survival under conditions of metabolic stress. Likewise, muscle-specific transgenic animals expressing a SNARK dominant-negative inactive mutant (SDN) had increased myonuclear apoptosis and activation of apoptotic mediators in muscle. Moreover, animals expressing SDN had severe, age-accelerated muscle atrophy and increased adiposity, consistent with sarcopenic obesity. Reduced SNARK activity, in vivo and in vitro, caused downregulation of the Rho kinase signaling pathway, a key mediator of cell survival. These findings reveal a critical role for SNARK in myocyte survival and the maintenance of muscle mass with age.

Authors

Sarah J. Lessard, Donato A. Rivas, Kawai So, Ho-Jin Koh, André Lima Queiroz, Michael F. Hirshman, Roger A. Fielding, Laurie J. Goodyear

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Abstract

Beckwith-Wiedemann syndrome (BWS) is a human stem cell disorder, and individuals with this disease have a substantially increased risk (~800-fold) of developing tumors. Epigenetic silencing of β2-spectrin (β2SP, encoded by SPTBN1), a SMAD adaptor for TGF-β signaling, is causally associated with BWS; however, a role of TGF-β deficiency in BWS-associated neoplastic transformation is unexplored. Here, we have reported that double-heterozygous Sptbn1+/– Smad3+/– mice, which have defective TGF-β signaling, develop multiple tumors that are phenotypically similar to those of BWS patients. Moreover, tumorigenesis-associated genes IGF2 and telomerase reverse transcriptase (TERT) were overexpressed in fibroblasts from BWS patients and TGF-β–defective mice. We further determined that chromatin insulator CCCTC-binding factor (CTCF) is TGF-β inducible and facilitates TGF-β–mediated repression of TERT transcription via interactions with β2SP and SMAD3. This regulation was abrogated in TGF-β–defective mice and BWS, resulting in TERT overexpression. Imprinting of the IGF2/H19 locus and the CDKN1C/KCNQ1 locus on chromosome 11p15.5 is mediated by CTCF, and this regulation is lost in BWS, leading to aberrant overexpression of growth-promoting genes. Therefore, we propose that loss of CTCF-dependent imprinting of tumor-promoting genes, such as IGF2 and TERT, results from a defective TGF-β pathway and is responsible at least in part for BWS-associated tumorigenesis as well as sporadic human cancers that are frequently associated with SPTBN1 and SMAD3 mutations.

Authors

Jian Chen, Zhi-Xing Yao, Jiun-Sheng Chen, Young Jin Gi, Nina M. Muñoz, Suchin Kundra, H. Franklin Herlong, Yun Seong Jeong, Alexei Goltsov, Kazufumi Ohshiro, Nipun A. Mistry, Jianping Zhang, Xiaoping Su, Sanaa Choufani, Abhisek Mitra, Shulin Li, Bibhuti Mishra, Jon White, Asif Rashid, Alan Yaoqi Wang, Milind Javle, Marta Davila, Peter Michaely, Rosanna Weksberg, Wayne L. Hofstetter, Milton J. Finegold, Jerry W. Shay, Keigo Machida, Hidekazu Tsukamoto, Lopa Mishra

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Abstract

Although defects in intestinal barrier function are a key pathogenic factor in patients with inflammatory bowel diseases (IBDs), the molecular pathways driving disease-specific alterations of intestinal epithelial cells (IECs) are largely unknown. Here, we addressed this issue by characterizing the transcriptome of IECs from IBD patients using a genome-wide approach. We observed disease-specific alterations in IECs with markedly impaired Rho-A signaling in active IBD patients. Localization of epithelial Rho-A was shifted to the cytosol in IBDs, and inflammation was associated with suppressed Rho-A activation due to reduced expression of the Rho-A prenylation enzyme geranylgeranyltransferase-I (GGTase-I). Functionally, we found that mice with conditional loss of Rhoa or the gene encoding GGTase-I, Pggt1b, in IECs exhibit spontaneous chronic intestinal inflammation with accumulation of granulocytes and CD4+ T cells. This phenotype was associated with cytoskeleton rearrangement and aberrant cell shedding, ultimately leading to loss of epithelial integrity and subsequent inflammation. These findings uncover deficient prenylation of Rho-A as a key player in the pathogenesis of IBDs. As therapeutic triggering of Rho-A signaling suppressed intestinal inflammation in mice with GGTase-I–deficient IECs, our findings suggest new avenues for treatment of epithelial injury and mucosal inflammation in IBD patients.

Authors

Rocío López-Posadas, Christoph Becker, Claudia Günther, Stefan Tenzer, Kerstin Amann, Ulrike Billmeier, Raja Atreya, Gionata Fiorino, Stefania Vetrano, Silvio Danese, Arif B. Ekici, Stefan Wirtz, Veronika Thonn, Alastair J.M. Watson, Cord Brakebusch, Martin Bergö, Markus F. Neurath, Imke Atreya

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Abstract

Worldwide, B cell non-Hodgkin lymphoma is the most common hematological malignancy and represents a substantial clinical problem. The molecular events that lead to B cell lymphoma are only partially defined. Here, we have provided evidence that deficiency of tetraspanin superfamily member CD37, which is important for B cell function, induces the development of B cell lymphoma. Mice lacking CD37 developed germinal center–derived B cell lymphoma in lymph nodes and spleens with a higher incidence than Bcl2 transgenic mice. We discovered that CD37 interacts with suppressor of cytokine signaling 3 (SOCS3); therefore, absence of CD37 drives tumor development through constitutive activation of the IL-6 signaling pathway. Moreover, animals deficient for both Cd37 and Il6 were fully protected against lymphoma development, confirming the involvement of the IL-6 pathway in driving tumorigenesis. Loss of CD37 on neoplastic cells in patients with diffuse large B cell lymphoma (DLBCL) directly correlated with activation of the IL-6 signaling pathway and with worse progression-free and overall survival. Together, this study identifies CD37 as a tumor suppressor that directly protects against B cell lymphomagenesis and provides a strong rationale for blocking the IL-6 pathway in patients with CD37 B cell malignancies as a possible therapeutic intervention.

Authors

Charlotte M. de Winde, Sharon Veenbergen, Ken H. Young, Zijun Y. Xu-Monette, Xiao-xiao Wang, Yi Xia, Kausar J. Jabbar, Michiel van den Brand, Alie van der Schaaf, Suraya Elfrink, Inge S. van Houdt, Marion J. Gijbels, Fons A.J. van de Loo, Miranda B. Bennink, Konnie M. Hebeda, Patricia J.T.A. Groenen, J. Han van Krieken, Carl G. Figdor, Annemiek B. van Spriel

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Abstract

The use of fibrinolytic agents to prevent new thrombus formation is limited by an increased risk of bleeding due to lysis of hemostatic clots that prevent hemorrhage in damaged blood vessels. We sought to develop an agent that provides thromboprophylaxis without carrying a significant risk of causing systemic fibrinolysis or disrupting hemostatic clots. We previously showed that platelet (PLT) α granule–delivered urokinase plasminogen activator (uPA) is highly effective in preventing thrombosis, while being associated with little systemic fibrinolysis or bleeding. Here, we generated a chimeric prodrug composed of a single-chain version of the variable region of an anti-αIIbβ3 mAb fused to a thrombin-activatable, low-molecular-weight pro-uPA (PLT/uPA-T). PLT/uPA-T recognizes human αIIbβ3 on both quiescent and activated platelets and is enzymatically activated specifically by thrombin. We found that this prodrug binds tightly to human platelets even after gel filtration, has a prolonged half-life in mice transgenic for human αIIb compared with that of uPA-T, and prevents clot formation in a microfluidic system. Importantly, in two murine injury models, PLT/uPA-T did not lyse preexisting clots, even when administration was delayed by as little as 10 minutes, while it concurrently prevented the development of nascent thrombi. Thus, PLT/uPA-T represents the prototype of a platelet-targeted thromboprophylactic agent that selectively targets nascent over preexisting thrombi.

Authors

Rudy E. Fuentes, Sergei Zaitsev, Hyun Sook Ahn, Vincent Hayes, M. Anna Kowalska, Michele P. Lambert, Yuhuan Wang, Donald L. Siegel, Daniel W. Bougie, Richard H. Aster, Daniel D. Myers, Victoria Stepanova, Douglas B. Cines, Vladimir R. Muzykantov, Mortimer Poncz

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Abstract

CYP24A1 (hereafter referred to as CYP24) enzymatic activity is pivotal in the inactivation of vitamin D metabolites. Basal renal and extrarenal CYP24 is usually low but is highly induced by its substrate 1,25-dihydroxyvitamin D. Unbalanced high and/or long-lasting CYP24 expression has been proposed to underlie diseases like chronic kidney disease, cancers, and psoriasis that otherwise should favorably respond to supplemental vitamin D. Using genetically modified mice, we have shown that renal phosphate wasting hypophosphatemic states arising from high levels of fibroblast growth factor 23 (FGF23) are also associated with increased renal Cyp24 expression, suggesting that elevated CYP24 activity is pivotal to the pathophysiology of these disorders. We therefore crossed 2 mouse strains, each with distinct etiology for high levels of circulating FGF23, onto a Cyp24-null background. Specifically, we evaluated Cyp24 deficiency in Hyp mice, the murine homolog of X-linked dominant hypophosphatemic rickets, and transgenic mice that overexpress a mutant FGF23 (FGF23R176Q) that is associated with the autosomal dominant form of hypophosphatemic rickets. Loss of Cyp24 in these murine models of human disease resulted in near-complete recovery of rachitic/osteomalacic bony abnormalities in the absence of any improvement in the serum biochemical profile. Moreover, treatment of Hyp and FGF23R1760-transgenic mice with the CYP24 inhibitor CTA102 also ameliorated their rachitic bones. Our results link CYP24 activity to the pathophysiology of FGF23-dependent renal phosphate wasting states and implicate pharmacologic CYP24 inhibition as a therapeutic adjunct for their treatment.

Authors

Xiuying Bai, Dengshun Miao, Sophia Xiao, Dinghong Qiu, René St-Arnaud, Martin Petkovich, Ajay Gupta, David Goltzman, Andrew C. Karaplis

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Abstract

The immunoregulatory cytokine macrophage migration inhibitory factor (MIF) is encoded in a functionally polymorphic locus that is linked to the susceptibility of autoimmune and infectious diseases. The MIF promoter contains a 4-nucleotide microsatellite polymorphism (–794 CATT) that repeats 5 to 8 times in the locus, with greater numbers of repeats associated with higher mRNA levels. Because there is no information about the transcriptional regulation of these common alleles, we used oligonucleotide affinity chromatography and liquid chromatography–mass spectrometry to identify nuclear proteins that interact with the –794 CATT5–8 site. An analysis of monocyte nuclear lysates revealed that the transcription factor ICBP90 (also known as UHRF1) is the major protein interacting with the MIF microsatellite. We found that ICBP90 is essential for MIF transcription from monocytes/macrophages, B and T lymphocytes, and synovial fibroblasts, and TLR-induced MIF transcription is regulated in an ICBP90- and –794 CATT5–8 length–dependent manner. Whole-genome transcription analysis of ICBP90 shRNA–treated rheumatoid synoviocytes uncovered a subset of proinflammatory and immune response genes that overlapped with those regulated by MIF shRNA. In addition, the expression levels of ICBP90 and MIF were correlated in joint synovia from patients with rheumatoid arthritis. These findings identify ICBP90 as a key regulator of MIF transcription and provide functional insight into the regulation of the polymorphic MIF locus.

Authors

Jie Yao, Lin Leng, Maor Sauler, Weiling Fu, Junsong Zheng, Yi Zhang, Xin Du, Xiaoqing Yu, Patty Lee, Richard Bucala

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Abstract

Recent studies have implicated chemokines in microglial activation and pathogenesis of neuropathic pain. C-X-C motif chemokine 13 (CXCL13) is a B lymphocyte chemoattractant that activates CXCR5. Using the spinal nerve ligation (SNL) model of neuropathic pain, we found that CXCL13 was persistently upregulated in spinal cord neurons after SNL, resulting in spinal astrocyte activation via CXCR5 in mice. shRNA-mediated inhibition of CXCL13 in the spinal cord persistently attenuated SNL-induced neuropathic pain. Interestingly, CXCL13 expression was suppressed by miR-186-5p, a microRNA that colocalized with CXCL13 and was downregulated after SNL. Spinal overexpression of miR-186-5p decreased CXCL13 expression, alleviating neuropathic pain. Furthermore, SNL induced CXCR5 expression in spinal astrocytes, and neuropathic pain was abrogated in Cxcr5–/– mice. CXCR5 expression induced by SNL was required for the SNL-induced activation of spinal astrocytes and microglia. Intrathecal injection of CXCL13 was sufficient to induce pain hypersensitivity and astrocyte activation via CXCR5 and ERK. Finally, intrathecal injection of CXCL13-activated astrocytes induced mechanical allodynia in naive mice. Collectively, our findings reveal a neuronal/astrocytic interaction in the spinal cord by which neuronally produced CXCL13 activates astrocytes via CXCR5 to facilitate neuropathic pain. Thus, miR-186-5p and CXCL13/CXCR5-mediated astrocyte signaling may be suitable therapeutic targets for neuropathic pain.

Authors

Bao-Chun Jiang, De-Li Cao, Xin Zhang, Zhi-Jun Zhang, Li-Na He, Chun-Hua Li, Wen-Wen Zhang, Xiao-Bo Wu, Temugin Berta, Ru-Rong Ji, Yong-Jing Gao

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Abstract

The nuclear pore complex protein NUP88 is frequently elevated in aggressive human cancers and correlates with reduced patient survival; however, it is unclear whether and how NUP88 overexpression drives tumorigenesis. Here, we show that mice overexpressing NUP88 are cancer prone and form intestinal tumors. To determine whether overexpression of NUP88 drives tumorigenesis, we engineered transgenic mice with doxycycline-inducible expression of Nup88. Surprisingly, NUP88 overexpression did not alter global nuclear transport, but was a potent inducer of aneuploidy and chromosomal instability. We determined that NUP88 and the nuclear transport factors NUP98 and RAE1 comprise a regulatory network that inhibits premitotic activity of the anaphase-promoting complex/cyclosome (APC/C). When overexpressed, NUP88 sequesters NUP98-RAE1 away from APC/CCDH1, triggering proteolysis of polo-like kinase 1 (PLK1), a tumor suppressor and multitasking mitotic kinase. Premitotic destruction of PLK1 disrupts centrosome separation, causing mitotic spindle asymmetry, merotelic microtubule-kinetochore attachments, lagging chromosomes, and aneuploidy. These effects were replicated by PLK1 insufficiency, indicating that PLK1 is responsible for the mitotic defects associated with NUP88 overexpression. These findings demonstrate that the NUP88-NUP98-RAE1-APC/CCDH1 axis contributes to aneuploidy and suggest that it may be deregulated in the initiating stages of a broad spectrum of human cancers.

Authors

Ryan M. Naylor, Karthik B. Jeganathan, Xiuqi Cao, Jan M. van Deursen

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Abstract

Osteoblast-derived VEGF is important for bone development and postnatal bone homeostasis. Previous studies have demonstrated that VEGF affects bone repair and regeneration; however, the cellular mechanisms by which it works are not fully understood. In this study, we investigated the functions of osteoblast-derived VEGF in healing of a bone defect. The results indicate that osteoblast-derived VEGF plays critical roles at several stages in the repair process. Using transgenic mice with osteoblast-specific deletion of Vegfa, we demonstrated that VEGF promoted macrophage recruitment and angiogenic responses in the inflammation phase, and optimal levels of VEGF were required for coupling of angiogenesis and osteogenesis in areas where repair occurs by intramembranous ossification. VEGF likely functions as a paracrine factor in this process because deletion of Vegfr2 in osteoblastic lineage cells enhanced osteoblastic maturation and mineralization. Furthermore, osteoblast- and hypertrophic chondrocyte–derived VEGF stimulated recruitment of blood vessels and osteoclasts and promoted cartilage resorption at the repair site during the periosteal endochondral ossification stage. Finally, osteoblast-derived VEGF stimulated osteoclast formation in the final remodeling phase of the repair process. These findings provide a basis for clinical strategies to improve bone regeneration and treat defects in bone healing.

Authors

Kai Hu, Bjorn R. Olsen

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Abstract

Deregulated protein and Ca2+ homeostasis underlie synaptic dysfunction and neurodegeneration in Huntington disease (HD); however, the factors that disrupt homeostasis are not fully understood. Here, we determined that expression of downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, is reduced in murine in vivo and in vitro HD models and in HD patients. DREAM downregulation was observed early after birth and was associated with endogenous neuroprotection. In the R6/2 mouse HD model, induced DREAM haplodeficiency or blockade of DREAM activity by chronic administration of the drug repaglinide delayed onset of motor dysfunction, reduced striatal atrophy, and prolonged life span. DREAM-related neuroprotection was linked to an interaction between DREAM and the unfolded protein response (UPR) sensor activating transcription factor 6 (ATF6). Repaglinide blocked this interaction and enhanced ATF6 processing and nuclear accumulation of transcriptionally active ATF6, improving prosurvival UPR function in striatal neurons. Together, our results identify a role for DREAM silencing in the activation of ATF6 signaling, which promotes early neuroprotection in HD.

Authors

José R. Naranjo, Hongyu Zhang, Diego Villar, Paz González, Xose M. Dopazo, Javier Morón-Oset, Elena Higueras, Juan C. Oliveros, María D. Arrabal, Angela Prieto, Pilar Cercós, Teresa González, Alicia De la Cruz, Juan Casado-Vela, Alberto Rábano, Carmen Valenzuela, Marta Gutierrez-Rodriguez, Jia-Yi Li, Britt Mellström

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Abstract

Renal erythropoietin-producing cells (REPCs) remain in the kidneys of patients with chronic kidney disease, but these cells do not produce sufficient erythropoietin in response to hypoxic stimuli. Treatment with HIF stabilizers rescues erythropoietin production in these cells, but the mechanisms underlying the decreased response of REPCs in fibrotic kidneys to anemic stimulation remain elusive. Here, we show that fibroblast-like FOXD1+ progenitor-derived kidney pericytes, which are characterized by the expression of α1 type I collagen and PDGFRβ, produce erythropoietin through HIF2α regulation but that production is repressed when these cells differentiate into myofibroblasts. DNA methyltransferases and erythropoietin hypermethylation are upregulated in myofibroblasts. Exposure of myofibroblasts to nanomolar concentrations of the demethylating agent 5-azacytidine increased basal expression and hypoxic induction of erythropoietin. Mechanistically, the profibrotic factor TGF-β1 induced hypermethylation and repression of erythropoietin in pericytes; these effects were prevented by 5-azacytidine treatment. These findings shed light on the molecular mechanisms underlying erythropoietin repression in kidney myofibroblasts and demonstrate that clinically relevant, nontoxic doses of 5-azacytidine can restore erythropoietin production and ameliorate anemia in the setting of kidney fibrosis in mice.

Authors

Yu-Ting Chang, Ching-Chin Yang, Szu-Yu Pan, Yu-Hsiang Chou, Fan-Chi Chang, Chun-Fu Lai, Ming-Hsuan Tsai, Huan-Lun Hsu, Ching-Hung Lin, Wen-Chih Chiang, Ming-Shiou Wu, Tzong-Shinn Chu, Yung-Ming Chen, Shuei-Liong Lin

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Abstract

The immune system has a powerful ability to recognize and kill cancer cells, but its function is often suppressed within tumors, preventing clearance of disease. Functionally diverse innate and adaptive cellular lineages either drive or constrain immune reactions within tumors. The transcription factor (TF) BACH2 regulates the differentiation of multiple innate and adaptive cellular lineages, but its role in controlling tumor immunity has not been elucidated. Here, we demonstrate that BACH2 is required to establish immunosuppression within tumors. Tumor growth was markedly impaired in Bach2-deficient mice and coincided with intratumoral activation of both innate and adaptive immunity. However, augmented tumor clearance in the absence of Bach2 was dependent upon the adaptive immune system. Analysis of tumor-infiltrating lymphocytes from Bach2-deficient mice revealed high frequencies of rapidly proliferating effector CD4+ and CD8+ T cells that expressed the inflammatory cytokine IFN-γ. Effector T cell activation coincided with a reduction in the frequency of intratumoral Foxp3+ Tregs. Mechanistically, BACH2 promoted tumor immunosuppression through Treg-mediated inhibition of intratumoral CD8+ T cells and IFN-γ. These findings demonstrate that BACH2 is a key component of the molecular program of tumor immunosuppression and identify therapeutic targets for the reversal of immunosuppression in cancer.

Authors

Rahul Roychoudhuri, Robert L. Eil, David Clever, Christopher A. Klebanoff, Madhusudhanan Sukumar, Francis M. Grant, Zhiya Yu, Gautam Mehta, Hui Liu, Ping Jin, Yun Ji, Douglas C. Palmer, Jenny H. Pan, Anna Chichura, Joseph G. Crompton, Shashank J. Patel, David Stroncek, Ena Wang, Francesco M. Marincola, Klaus Okkenhaug, Luca Gattinoni, Nicholas P. Restifo

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Abstract

Colon tumors arise in a stepwise fashion from either discrete genetic perturbations or epigenetic dysregulation. To uncover the key epigenetic regulators that drive colon cancer growth, we used a CRISPR loss-of-function screen and identified a number of essential genes, including the bromodomain and extraterminal (BET) protein BRD4. We found that BRD4 is critical for colon cancer proliferation, and its knockdown led to differentiation effects in vivo. JQ1, a BET inhibitor, preferentially reduced growth in a subset of epigenetically dysregulated colon cancers characterized by the CpG island methylator phenotype (CIMP). Integrated transcriptomic and genomic analyses defined a distinct superenhancer in CIMP+ colon cancers that regulates cMYC transcription. We found that the long noncoding RNA colon cancer–associated transcript 1 (CCAT1) is transcribed from this superenhancer and is exquisitely sensitive to BET inhibition. Concordantly, cMYC transcription and cell growth were tightly correlated with the presence of CCAT1 RNA in a variety of tumor types. Taken together, we propose that CCAT1 is a clinically tractable biomarker for identifying patients who are likely to benefit from BET inhibitors.

Authors

Mark L. McCleland, Kathryn Mesh, Edward Lorenzana, Vivek S. Chopra, Ehud Segal, Colin Watanabe, Benjamin Haley, Oleg Mayba, Murat Yaylaoglu, Florian Gnad, Ron Firestein

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Abstract

The nature and role of the intestinal leukocytes in necrotizing enterocolitis (NEC), a severe disease affecting premature infants, remain unknown. We now show that the intestine in mouse and human NEC is rich in lymphocytes that are required for NEC development, as recombination activating gene 1–deficient (Rag1–/–) mice were protected from NEC and transfer of intestinal lymphocytes from NEC mice into naive mice induced intestinal inflammation. The intestinal expression of the lipopolysaccharide receptor TLR4, which is higher in the premature compared with full-term human and mouse intestine, is required for lymphocyte influx through TLR4-mediated upregulation of CCR9/CCL25 signaling. TLR4 also mediates a STAT3-dependent polarization toward increased proinflammatory CD3+CD4+IL-17+ and reduced tolerogenic Foxp3+ Treg lymphocytes (Tregs). Th17 lymphocytes were required for NEC development, as inhibition of STAT3 or IL-17 receptor signaling attenuated NEC in mice, while IL-17 release impaired enterocyte tight junctions, increased enterocyte apoptosis, and reduced enterocyte proliferation, leading to NEC. Importantly, TLR4-dependent Th17 polarization could be reversed by the enteral administration of retinoic acid, which induced Tregs and decreased NEC severity. These findings identify an important role for proinflammatory lymphocytes in NEC development via intestinal epithelial TLR4 that could be reversed through dietary modification.

Authors

Charlotte E. Egan, Chhinder P. Sodhi, Misty Good, Joyce Lin, Hongpeng Jia, Yukihiro Yamaguchi, Peng Lu, Congrong Ma, Maria F. Branca, Samantha Weyandt, William B. Fulton, Diego F. Niño, Thomas Prindle Jr., John A. Ozolek, David J. Hackam

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Abstract

Evolutionarily conserved receptor tyrosine kinase–like orphan receptor-1 and -2 (ROR1/2) are considered distinct receptors for Wnt5a and are implicated in noncanonical Wnt signaling in organogenesis and cancer metastasis. We found that Wnt5a enhanced proliferation and migration of chronic lymphocytic leukemia (CLL) cells and that these effects were blocked by the humanized anti-ROR1 mAb cirmtuzumab (UC-961). Treatment of CLL cells with Wnt5a induced ROR1 to oligomerize with ROR2 and recruit guanine exchange factors (GEFs), which activated Rac1 and RhoA; siRNA-mediated silencing of either ROR1 or ROR2 or treatment with UC-961 inhibited these effects. Using the ROR1-deficient CLL cell line MEC1, we demonstrated that ectopic ROR1 expression induced ROR1/ROR2 heterooligomers, which recruited GEFs, and enhanced proliferation, cytokine-directed migration, and engraftment potential of MEC1 cells in immune-deficient mice. Notably, treatment with UC-961 inhibited engraftment of ROR1+ leukemia cells in immune-competent ROR1-transgenic mice. Molecular analysis revealed that the extracellular Kringle domain is required for ROR1/ROR2 heterooligomerization and the cysteine-rich domain or intracellular proline-rich domain is required for Wnt5a-induced recruitment of GEFs to ROR1/ROR2. This study identifies an interaction between ROR1 and ROR2 that is required for Wnt5a signaling that promotes leukemia chemotaxis and proliferation.

Authors

Jian Yu, Liguang Chen, Bing Cui, George F. Widhopf II, Zhouxin Shen, Rongrong Wu, Ling Zhang, Suping Zhang, Steven P. Briggs, Thomas J. Kipps

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Abstract

Colorectal cancer metastasis to the liver is a major cause of cancer-related death; however, the genes and pathways that govern this metastatic colonization event remain poorly characterized. Here, using a large-scale in vivo RNAi screen, we identified liver and red blood cell pyruvate kinase (PKLR) as a driver of metastatic liver colonization. PKLR expression was increased in liver metastases as well as in primary colorectal tumors of patients with metastatic disease. Evaluation of a murine liver colonization model revealed that PKLR promotes cell survival in the tumor core during conditions of high cell density and oxygen deprivation by increasing glutathione, the primary endogenous antioxidant. PKLR negatively regulated the glycolytic activity of PKM2, the major pyruvate kinase isoenzyme known to regulate cellular glutathione levels. Glutathione is critical for metastasis, and we determined that the rate-limiting enzyme of glutathione synthesis, GCLC, becomes overexpressed in patient liver metastases, promotes cell survival under hypoxic and cell-dense conditions, and mediates metastatic liver colonization. RNAi-mediated inhibition of glutathione synthesis impaired survival of multiple colon cancer cell lines, and pharmacological targeting of this metabolic pathway reduced colonization in a primary patient-derived xenograft model. Our findings highlight the impact of metabolic reprogramming within the niche as metastases progress and suggest clinical potential for targeting this pathway in colorectal cancer.

Authors

Alexander Nguyen, Jia Min Loo, Rohit Mital, Ethan M. Weinberg, Fung Ying Man, Zhaoshi Zeng, Philip B. Paty, Leonard Saltz, Yelena Y. Janjigian, Elisa de Stanchina, Sohail F. Tavazoie

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Abstract

Tumor cells frequently escape from CD8+ T cell recognition by abrogating MHC-I antigen presentation. Deficiency in processing components, like the transporter associated with antigen processing (TAP), results in strongly decreased surface display of peptide/MHC-I complexes. We previously identified a class of hidden self-antigens known as T cell epitopes associated with impaired peptide processing (TEIPP), which emerge on tumor cells with such processing defects. In the present study, we analyzed thymus selection and peripheral behavior of T cells with specificity for the prototypic TEIPP antigen, the “self” TRH4 peptide/Db complex. TEIPP T cells were efficiently selected in the thymus, egressed with a naive phenotype, and could be exploited for immunotherapy against immune-escaped, TAP-deficient tumor cells expressing low levels of MHC-I (MHC-Ilo). In contrast, overt thymus deletion and functionally impaired TEIPP T cells were observed in mice deficient for TAP1 due to TEIPP antigen presentation on all body cells in these mice. Our results strongly support the concept that TEIPPs derive from ubiquitous, nonmutated self-antigens and constitute a class of immunogenic neoantigens that are unmasked during tumor immune evasion. These data suggest that TEIPP-specific CD8+ T cells are promising candidates in the treatment of tumors that have escaped from conventional immunotherapies.

Authors

Elien M. Doorduijn, Marjolein Sluijter, Bianca J. Querido, Cláudia C. Oliveira, Adnane Achour, Ferry Ossendorp, Sjoerd H. van der Burg, Thorbald van Hall

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Abstract

Systemic inflammation causes malaise and general feelings of discomfort. This fundamental aspect of the sickness response reduces the quality of life for people suffering from chronic inflammatory diseases and is a nuisance during mild infections like common colds or the flu. To investigate how inflammation is perceived as unpleasant and causes negative affect, we used a behavioral test in which mice avoid an environment that they have learned to associate with inflammation-induced discomfort. Using a combination of cell-type–specific gene deletions, pharmacology, and chemogenetics, we found that systemic inflammation triggered aversion through MyD88-dependent activation of the brain endothelium followed by COX1-mediated cerebral prostaglandin E2 (PGE2) synthesis. Further, we showed that inflammation-induced PGE2 targeted EP1 receptors on striatal dopamine D1 receptor–expressing neurons and that this signaling sequence induced aversion through GABA-mediated inhibition of dopaminergic cells. Finally, we demonstrated that inflammation-induced aversion was not an indirect consequence of fever or anorexia but that it constituted an independent inflammatory symptom triggered by a unique molecular mechanism. Collectively, these findings demonstrate that PGE2-mediated modulation of the dopaminergic motivational circuitry is a key mechanism underlying the negative affect induced by inflammation.

Authors

Michael Fritz, Anna M. Klawonn, Anna Nilsson, Anand Kumar Singh, Joanna Zajdel, Daniel Björk Wilhelms, Michael Lazarus, Andreas Löfberg, Maarit Jaarola, Unn Örtegren Kugelberg, Timothy R. Billiar, David J. Hackam, Chhinder P. Sodhi, Matthew D. Breyer, Johan Jakobsson, Markus Schwaninger, Günther Schütz, Jan Rodriguez Parkitna, Clifford B. Saper, Anders Blomqvist, David Engblom

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Abstract

In vivo protection by antimicrobial neutralizing Abs can require the contribution of effector functions mediated by Fc-Fcγ receptor (Fc-FcγR) interactions for optimal efficacy. In influenza, broadly neutralizing anti-hemagglutinin (anti-HA) stalk mAbs require Fc-FcγR interactions to mediate in vivo protection, but strain-specific anti-HA head mAbs do not. Whether this rule applies only to anti-stalk Abs or is applicable to any broadly neutralizing Ab (bNAb) against influenza is unknown. Here, we characterized the contribution of Fc-FcγR interactions during in vivo protection for a panel of 13 anti-HA mAbs, including bNAbs and non-neutralizing Abs, against both the stalk and head domains. All classes of broadly binding anti-HA mAbs required Fc-FcγR interactions to provide protection in vivo, including those mAbs that bind the HA head and those that do not neutralize virus in vitro. Further, a broadly neutralizing anti-neuraminidase (anti-NA) mAb also required FcγRs to provide protection in vivo, but a strain-specific anti-NA mAb did not. Thus, these findings suggest that the breadth of reactivity of anti-influenza Abs, regardless of their epitope, necessitates interactions with FcγRs on effector cell populations to mediate in vivo protection. These findings will guide the design of antiviral Ab therapeutics and inform vaccine design to elicit Abs with optimal binding properties and effector functions.

Authors

David J. DiLillo, Peter Palese, Patrick C. Wilson, Jeffrey V. Ravetch

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Abstract

BACKGROUND. Arthrogryposis, defined as congenital joint contractures in 2 or more body areas, is a clinical sign rather than a specific disease diagnosis. To date, more than 400 different disorders have been described that present with arthrogryposis, and variants of more than 220 genes have been associated with these disorders; however, the underlying molecular etiology remains unknown in the considerable majority of these cases.

METHODS. We performed whole exome sequencing (WES) of 52 patients with clinical presentation of arthrogryposis from 48 different families.

RESULTS. Affected individuals from 17 families (35.4%) had variants in known arthrogryposis-associated genes, including homozygous variants of cholinergic γ nicotinic receptor (CHRNG, 6 subjects) and endothelin converting enzyme–like 1 (ECEL1, 4 subjects). Deleterious variants in candidate arthrogryposis-causing genes (fibrillin 3 [FBN3], myosin IXA [MYO9A], and pleckstrin and Sec7 domain containing 3 [PSD3]) were identified in 3 families (6.2%). Moreover, in 8 families with a homozygous mutation in an arthrogryposis-associated gene, we identified a second locus with either a homozygous or compound heterozygous variant in a candidate gene (myosin binding protein C, fast type [MYBPC2] and vacuolar protein sorting 8 [VPS8], 2 families, 4.2%) or in another disease-associated genes (6 families, 12.5%), indicating a potential mutational burden contributing to disease expression.

CONCLUSION. In 58.3% of families, the arthrogryposis manifestation could be explained by a molecular diagnosis; however, the molecular etiology in subjects from 20 families remained unsolved by WES. Only 5 of these 20 unrelated subjects had a clinical presentation consistent with amyoplasia; a phenotype not thought to be of genetic origin. Our results indicate that increased use of genome-wide technologies will provide opportunities to better understand genetic models for diseases and molecular mechanisms of genetically heterogeneous disorders, such as arthrogryposis.

FUNDING. This work was supported in part by US National Human Genome Research Institute (NHGRI)/National Heart, Lung, and Blood Institute (NHLBI) grant U54HG006542 to the Baylor-Hopkins Center for Mendelian Genomics, and US National Institute of Neurological Disorders and Stroke (NINDS) grant R01NS058529 to J.R. Lupski.

Authors

Yavuz Bayram, Ender Karaca, Zeynep Coban Akdemir, Elif Ozdamar Yilmaz, Gulsen Akay Tayfun, Hatip Aydin, Deniz Torun, Sevcan Tug Bozdogan, Alper Gezdirici, Sedat Isikay, Mehmed M. Atik, Tomasz Gambin, Tamar Harel, Ayman W. El-Hattab, Wu-Lin Charng, Davut Pehlivan, Shalini N. Jhangiani, Donna M. Muzny, Ali Karaman, Tamer Celik, Ozge Ozalp Yuregir, Timur Yildirim, Ilhan A. Bayhan, Eric Boerwinkle, Richard A. Gibbs, Nursel Elcioglu, Beyhan Tuysuz, James R. Lupski

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Abstract

Overexpression of FGF23 results in hypophosphatemic rickets, which is characterized by renal phosphate wasting, inappropriately low circulating levels of the active form of vitamin D, and skeletal abnormalities. The precise mechanisms of how excess FGF23 leads to hypophosphatemic rickets are not clear. In this issue of the JCI, Bai and colleagues demonstrate that deletion or inhibition of CYP24A1, which initiates degradation of the active form of vitamin D, ameliorates skeletal abnormalities in two mouse models of hypophosphatemic rickets. While this work supports an important role for excess CYP24A1 activity in the pathogenesis of FGF23-mediated hypophosphatemic rickets, more work will need to be done before CYP24A1 inhibition can be integrated into the management of patients living with these diseases.

Authors

Valentin David, Myles Wolf

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Abstract

This position statement originated from a working group meeting convened on April 15, 2015, by the NHLBI and incorporates follow-up contributions by the participants as well as other thought leaders subsequently consulted, who together represent research fields relevant to all branches of the NIH. The group was deliberately composed not only of individuals with a current research emphasis in the glycosciences, but also of many experts from other fields, who evinced a strong interest in being involved in the discussions. The original goal was to discuss the value of creating centers of excellence for training the next generation of biomedical investigators in the glycosciences. A broader theme that emerged was the urgent need to bring the glycosciences back into the mainstream of biology by integrating relevant education into the curricula of medical, graduate, and postgraduate training programs, thus generating a critical sustainable workforce that can advance the much-needed translation of glycosciences into a more complete understanding of biology and the enhanced practice of medicine.

Authors

Peter Agre, Carolyn Bertozzi, Mina Bissell, Kevin P. Campbell, Richard D. Cummings, Umesh R. Desai, Mary Estes, Terence Flotte, Guy Fogleman, Fred Gage, David Ginsburg, Jeffrey I. Gordon, Gerald Hart, Vincent Hascall, Laura Kiessling, Stuart Kornfeld, John Lowe, John Magnani, Lara K. Mahal, Ruslan Medzhitov, Richard J. Roberts, Robert Sackstein, Rita Sarkar, Ronald Schnaar, Nancy Schwartz, Ajit Varki, David Walt, Irving Weissman

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Abstract

Although the cognitive and biological characteristics of Alzheimer’s disease (AD) are well known and mouse models of AD are available, current treatments for AD-related cognitive deficits have quite limited efficacy. The development of tasks with cross-species validity may enable better prediction of the efficacy of potential new treatments. In this issue of the JCI, Possin et al. present a virtual version of the Morris water maze (a common test of spatial learning and memory for rodents) that is designed for use with humans. The authors tested a mouse model of AD (transgenic mice expressing human amyloid precursor protein [hAPP]) and patients in the earlier mild cognitive impairment (MCI) stage of AD in their respective versions of the maze. Using novel statistical methods, they detected similar deficits across species, providing support for the hAPP model and use of the virtual water maze. Importantly, this work enabled recommendations for appropriate sample sizes when developing potential therapeutics for AD.

Authors

Kerin K. Higa, Jared W. Young, Mark A. Geyer

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Abstract

Cancer immunotherapy in which cytotoxic T cells (CTLs) target tumor-specific antigens complexed to MHC-I molecules has been used successfully for several types of cancer; however, MHC-I is frequently downregulated in tumors, resulting in CTL evasion. Recently, it has been shown that MHC-Ilo tumors produce a set of T cell epitopes associated with impaired peptide processing (TEIPP) that have potential to be exploited for immunotherapy. TEIPP-specific CTLs recognize tumors defective in antigen presentation machinery (APM) but not those with intact APM. In this issue of the JCI, Doorduljn et al. evaluated thymus selection and peripheral behavior of TEIPP-specific T cells, using a unique T cell receptor (TCR) transgenic mouse model. They demonstrated that TEIPP-specific T cells in TAP-deficient mice have largely been deleted by central tolerance, while the same T cells in WT mice are naive and sustained. The results of this study suggest that TIEPPs have potential to be successful targets for elimination of MHC-Ilo tumors.

Authors

Rolf Kiessling

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February 2016

126 2 cover

February 2016 Issue

On the cover:
Metabolic reprogramming in the metastatic niche

This month’s cover image shows a liver tumor nodule following injection of colorectal cancer cells depleted of PKLR (liver and red blood cell pyruvate kinase), with staining for tumor cells (luciferase, green), apoptotic cells (cleaved caspase-3, red), and nuclei (DAPI, blue). On page 681, Nguyen et al. demonstrate that liver metastases of colorectal cancer are driven by pyruvate kinase isozyme expression.

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Jci tm 2016 02

February 2016 JCI This Month

JCI This Month is a digest of the research, reviews, and other features published each month.

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Review Series - More

HIV

Series edited by Robert F. Siliciano

The development of combination antiretroviral therapy (ART) in the mid-1990s initially raised hopes that HIV was a curable disease; however, further studies revealed that the virus persists, even in patients with undetectable levels of HIV in their plasma. Resting CD4+ T cells harbor stably integrated viral genomes that can produce infectious virus following T cell activation. Importantly, treatment interruption leads to a rapid recrudescence of infection from this latent reservoir, usually within 2 to 3 weeks. Several distinct areas of HIV research are now focused on the development of strategies to precent the latent reservoir from replicating or to eliminate it entirely. Reviews in this series detail progress in our understanding of the molecular and cellular mechanisms of viral latency, efforts to accurately assess the size and composition of the latent viral reservoir, the characterization and development of HIV-targeted broadly neutralizing antibodies and cytolytic T lymphocytes, as well as animal models for the study of HIV latency and therapeutic strategies.

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