Research Article
Shared signaling networks active in B cells isolated from genetically distinct mouse models of lupus
Abstract
Though B cells play key roles in lupus pathogenesis, the molecular circuitry and its dysregulation in these cells as disease evolves remain poorly understood. To address this, a comprehensive scan of multiple signaling axes using multiplexed Western blotting was undertaken in several different murine lupus strains. PI3K/AKT/mTOR (mTOR, mammalian target of rapamycin), MEK1/Erk1/2, p38, NF-κB, multiple Bcl-2 family members, and cell-cycle molecules were observed to be hyperexpressed in lupus B cells in an age-dependent and lupus susceptibility gene–dose–dependent manner. Therapeutic targeting of the AKT/mTOR axis using a rapamycin (sirolimus) derivative ameliorated the serological, cellular, and pathological phenotypes associated with lupus. Surprisingly, the targeting of this axis was associated with the crippling of several other signaling axes. These studies reveal that lupus pathogenesis is contingent upon the activation of an elaborate network of signaling cascades that is shared among genetically distinct mouse models and raise hope that targeting pivotal nodes in these networks may offer therapeutic benefit.
Authors
Tianfu Wu, Xiangmei Qin, Zoran Kurepa, Kirthi Raman Kumar, Kui Liu, Hasna Kanta, Xin J. Zhou, Anne B. Satterthwaite, Laurie S. Davis, Chandra Mohan
Abstract
Genetic variants in the gene encoding for transcription factor-7–like 2 (TCF7L2) have been associated with type 2 diabetes (T2D) and impaired β cell function, but the mechanisms have remained unknown. We therefore studied prospectively the ability of common variants in TCF7L2 to predict future T2D and explored the mechanisms by which they would do this. Scandinavian subjects followed for up to 22 years were genotyped for 3 SNPs (rs7903146, rs12255372, and rs10885406) in TCF7L2, and a subset of them underwent extensive metabolic studies. Expression of TCF7L2 was related to genotype and metabolic parameters in human islets. The CT/TT genotypes of SNP rs7903146 strongly predicted future T2D in 2 independent cohorts (Swedish and Finnish). The risk T allele was associated with impaired insulin secretion, incretin effects, and enhanced rate of hepatic glucose production. TCF7L2 expression in human islets was increased 5-fold in T2D, particularly in carriers of the TT genotype. Overexpression of TCF7L2 in human islets reduced glucose-stimulated insulin secretion. In conclusion, the increased risk of T2D conferred by variants in TCF7L2 involves the enteroinsular axis, enhanced expression of the gene in islets, and impaired insulin secretion.
Authors
Valeriya Lyssenko, Roberto Lupi, Piero Marchetti, Silvia Del Guerra, Marju Orho-Melander, Peter Almgren, Marketa Sjögren, Charlotte Ling, Karl-Fredrik Eriksson, υsa-Linda Lethagen, Rita Mancarella, Göran Berglund, Tiinamaija Tuomi, Peter Nilsson, Stefano Del Prato, Leif Groop
Abstract
Noonan syndrome (NS) is an autosomal dominant disorder characterized by a wide spectrum of defects, which most frequently include proportionate short stature, craniofacial anomalies, and congenital heart disease (CHD). NS is the most common nonchromosomal cause of CHD, and 80%–90% of NS patients have cardiac involvement. Mutations within the protein tyrosine phosphatase Src homology region 2, phosphatase 2 (SHP2) are responsible for approximately 50% of the cases of NS with cardiac involvement. To understand the developmental stage– and cell type–specific consequences of the NS SHP2 gain-of-function mutation, Q79R, we generated transgenic mice in which the mutated protein was expressed during gestation or following birth in cardiomyocytes. Q79R SHP2 embryonic hearts showed altered cardiomyocyte cell cycling, ventricular noncompaction, and ventricular septal defects, while, in the postnatal cardiomyocyte, Q79R SHP2 expression was completely benign. Fetal expression of Q79R led to the specific activation of the ERK1/2 pathway, and breeding of the Q79R transgenics into ERK1/2-null backgrounds confirmed the pathway’s necessity and sufficiency in mediating mutant SHP2’s effects. Our data establish the developmental stage–specific effects of Q79R cardiac expression in NS; show that ablation of subsequent ERK1/2 activation prevents the development of cardiac abnormalities; and suggest that ERK1/2 modulation could have important implications for developing therapeutic strategies in CHD.
Authors
Tomoki Nakamura, Melissa Colbert, Maike Krenz, Jeffery D. Molkentin, Harvey S. Hahn, Gerald W. Dorn II, Jeffrey Robbins
Abstract
In vitro studies indicate that binding of talin to the β3 integrin cytoplasmic domain (tail) results in integrin αIIbβ3 (GPIIb–IIIa) activation. Here we tested the importance of talin binding for integrin activation in vivo and its biological significance by generating mice harboring point mutations in the β3 tail. We introduced a β3(Y747A) substitution that disrupts the binding of talin, filamin, and other cytoplasmic proteins and a β3(L746A) substitution that selectively disrupts interactions only with talin. Platelets from animals homozygous for each mutation showed impaired agonist-induced fibrinogen binding and platelet aggregation, providing proof that inside-out signals that activate αIIbβ3 require binding of talin to the β3 tail. β3(L746A) mice were resistant to both pulmonary thromboembolism and to ferric chloride–induced thrombosis of the carotid artery. Pathological bleeding, measured by the presence of fecal blood and development of anemia, occurred in 53% of β3(Y747A) and virtually all β3-null animals examined. Remarkably, less than 5% of β3(L746A) animals exhibited this form of bleeding. These results establish that αIIbβ3 activation in vivo is dependent on the interaction of talin with the β3 integrin cytoplasmic domain. Furthermore, they suggest that modulation of β3 integrin–talin interactions may provide an attractive target for antithrombotics and result in a reduced risk of pathological bleeding.
Authors
Brian G. Petrich, Per Fogelstrand, Anthony W. Partridge, Nima Yousefi, Ararat J. Ablooglu, Sanford J. Shattil, Mark H. Ginsberg
Abstract
We hypothesized that overexpression of PDGF-BB in colorectal cancer (CRC) and pancreatic cancer cells would result in increased pericyte coverage of ECs in vivo, rendering the tumor vasculature more resistant to antiangiogenic therapy. We stably transfected the cDNA for the PDGF-B into HT-29 human CRC and FG human pancreatic cancer cells. Surprisingly, when HT-29 or FG parental and transfected cells were injected into mice (subcutaneously and orthotopically), we observed marked inhibition of tumor growth in the PDGF-BB–overexpressing clones. In the PDGF-BB–overexpressing tumors, we observed an increase in pericyte coverage of ECs. Treatment of PDGF-BB–overexpressing tumors with imatinib mesylate (PDGFR inhibitor) resulted in increased growth and decreased total pericyte content compared with those in untreated PDGF-BB–overexpressing tumors. In vitro studies demonstrated the ability of VSMCs to inhibit EC proliferation by approximately 50%. These data show that increasing the pericyte content of the tumor microenvironment inhibits the growth of angiogenesis-dependent tumors. Single-agent therapy targeting PDGF receptor must be used with caution in tumors when PDGFR is not the target on the tumor cell itself.
Authors
Marya F. McCarty, Ray J. Somcio, Oliver Stoeltzing, Jane Wey, Fan Fan, Wenbiao Liu, Corazon Bucana, Lee M. Ellis
Abstract
Obesity, the metabolic syndrome, and type 2 diabetes mellitus (T2DM) are major global health problems. Insulin resistance is frequently present in these disorders, but the causes and effects of such resistance are unknown. Here, we generated mice with muscle-specific knockout of the major murine atypical PKC (aPKC), PKC-λ, a postulated mediator for insulin-stimulated glucose transport. Glucose transport and translocation of glucose transporter 4 (GLUT4) to the plasma membrane were diminished in muscles of both homozygous and heterozygous PKC-λ knockout mice and were accompanied by systemic insulin resistance; impaired glucose tolerance or diabetes; islet β cell hyperplasia; abdominal adiposity; hepatosteatosis; elevated serum triglycerides, FFAs, and LDL-cholesterol; and diminished HDL-cholesterol. In contrast to the defective activation of muscle aPKC, insulin signaling and actions were intact in muscle, liver, and adipocytes. These findings demonstrate the importance of aPKC in insulin-stimulated glucose transport in muscles of intact mice and show that insulin resistance and resultant hyperinsulinemia owing to a specific defect in muscle aPKC is sufficient to induce abdominal obesity and other lipid abnormalities of the metabolic syndrome and T2DM. These findings are particularly relevant because humans who have obesity, impaired glucose tolerance, and T2DM reportedly have defective activation and/or diminished levels of muscle aPKC.
Authors
Robert V. Farese, Mini P. Sajan, Hong Yang, Pengfei Li, Steven Mastorides, William R. Gower Jr., Sonali Nimal, Cheol Soo Choi, Sheene Kim, Gerald I. Shulman, C. Ronald Kahn, Ursula Braun, Michael Leitges
Abstract
Hypothalamic AMP-activated protein kinase (AMPK) has been suggested to act as a key sensing mechanism, responding to hormones and nutrients in the regulation of energy homeostasis. However, the precise neuronal populations and cellular mechanisms involved are unclear. The effects of long-term manipulation of hypothalamic AMPK on energy balance are also unknown. To directly address such issues, we generated POMCα2KO and AgRPα2KO mice lacking AMPKα2 in proopiomelanocortin– (POMC-) and agouti-related protein–expressing (AgRP-expressing) neurons, key regulators of energy homeostasis. POMCα2KO mice developed obesity due to reduced energy expenditure and dysregulated food intake but remained sensitive to leptin. In contrast, AgRPα2KO mice developed an age-dependent lean phenotype with increased sensitivity to a melanocortin agonist. Electrophysiological studies in AMPKα2-deficient POMC or AgRP neurons revealed normal leptin or insulin action but absent responses to alterations in extracellular glucose levels, showing that glucose-sensing signaling mechanisms in these neurons are distinct from those pathways utilized by leptin or insulin. Taken together with the divergent phenotypes of POMCα2KO and AgRPα2KO mice, our findings suggest that while AMPK plays a key role in hypothalamic function, it does not act as a general sensor and integrator of energy homeostasis in the mediobasal hypothalamus.
Authors
Marc Claret, Mark A. Smith, Rachel L. Batterham, Colin Selman, Agharul I. Choudhury, Lee G.D. Fryer, Melanie Clements, Hind Al-Qassab, Helen Heffron, Allison W. Xu, John R. Speakman, Gregory S. Barsh, Benoit Viollet, Sophie Vaulont, Michael L.J. Ashford, David Carling, Dominic J. Withers
Abstract
Because of the paucity of known self lipid–reactive ligands for NKT cells, interactions among distinct NKT cell subsets as well as immune consequences following recognition of self glycolipids have not previously been investigated. Here we examined cellular interactions and subsequent immune regulatory mechanism following recognition of sulfatide, a self-glycolipid ligand for a subset of CD1d-restricted type II NKT cells. Using glycolipid/CD1d tetramers and cytokine responses, we showed that activation of sulfatide-reactive type II NKT cells and plasmacytoid DCs caused IL-12– and MIP-2–dependent recruitment of type I, or invariant, NKT (iNKT) cells into mouse livers. These recruited iNKT cells were anergic and prevented concanavalin A–induced (ConA-induced) hepatitis by specifically blocking effector pathways, including the cytokine burst and neutrophil recruitment that follow ConA injection. Hepatic DCs from IL-12+/+ mice, but not IL-12–/– mice, adoptively transferred anergy in recipients; thus, IL-12 secretion by DCs enables them to induce anergy in iNKT cells. Our data reveal what we believe to be a novel mechanism in which interactions among type II NKT cells and hepatic DCs result in regulation of iNKT cell activity that can be exploited for intervention in inflammatory diseases, including autoimmunity and asthma.
Authors
Ramesh C. Halder, Carlos Aguilera, Igor Maricic, Vipin Kumar
Abstract
SWAP-70–like adapter of T cells (SLAT) is a novel guanine nucleotide exchange factor for Rho GTPases that is upregulated in Th2 cells, but whose physiological function is unclear. We show that SLAT–/– mice displayed a developmental defect at one of the earliest stages of thymocyte differentiation, the double-negative 1 (DN1) stage, leading to decreased peripheral T cell numbers. SLAT–/– peripheral CD4+ T cells demonstrated impaired TCR/CD28-induced proliferation and IL-2 production, which was rescued by the addition of exogenous IL-2. Importantly, SLAT–/– mice were grossly impaired in their ability to mount not only Th2, but also Th1-mediated lung inflammatory responses, as evidenced by reduced airway neutrophilia and eosinophilia, respectively. Levels of Th1 and Th2 cytokine in the lungs were also markedly reduced, paralleling the reduction in pulmonary inflammation. This defect in mounting Th1/Th2 responses, which was also evident in vitro, was traced to a severe reduction in Ca2+ mobilization from ER stores, which consequently led to defective TCR/CD28-induced translocation of nuclear factor of activated T cells 1/2 (NFATc1/2). Thus, SLAT is required for thymic DN1 cell expansion, T cell activation, and Th1 and Th2 inflammatory responses.
Authors
Stéphane Bécart, Céline Charvet, Ann J. Canonigo Balancio, Carl De Trez, Yoshihiko Tanaka, Wei Duan, Carl Ware, Michael Croft, Amnon Altman
Abstract
Recent reports have challenged the notion that retroviruses and retroviral vectors integrate randomly into the host genome. These reports pointed to a strong bias toward integration in and near gene coding regions and, for gammaretroviral vectors, around transcription start sites. Here, we report the results obtained from a large-scale mapping of 572 retroviral integration sites (RISs) isolated from cells of 9 patients with X-linked SCID (SCID-X1) treated with a retrovirus-based gene therapy protocol. Our data showed that two-thirds of insertions occurred in or very near to genes, of which more than half were highly expressed in CD34+ progenitor cells. Strikingly, one-fourth of all integrations were clustered as common integration sites (CISs). The highly significant incidence of CISs in circulating T cells and the nature of their locations indicate that insertion in many gene loci has an influence on cell engraftment, survival, and proliferation. Beyond the observed cases of insertional mutagenesis in 3 patients, these data help to elucidate the relationship between vector insertion and long-term in vivo selection of transduced cells in human patients with SCID-X1.
Authors
Annette Deichmann, Salima Hacein-Bey-Abina, Manfred Schmidt, Alexandrine Garrigue, Martijn H. Brugman, Jingqiong Hu, Hanno Glimm, Gabor Gyapay, Bernard Prum, Christopher C. Fraser, Nicolas Fischer, Kerstin Schwarzwaelder, Maria-Luise Siegler, Dick de Ridder, Karin Pike-Overzet, Steven J. Howe, Adrian J. Thrasher, Gerard Wagemaker, Ulrich Abel, Frank J.T. Staal, Eric Delabesse, Jean-Luc Villeval, Bruce Aronow, Christophe Hue, Claudia Prinz, Manuela Wissler, Chuck Klanke, Jean Weissenbach, Ian Alexander, Alain Fischer, Christof von Kalle, Marina Cavazzana-Calvo
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ISSN: 0021-9738 (print), 1558-8238 (online)