Research Article
Abstract
Constitutive activation of NF-κB is a frequent event in human cancers, playing important roles in cancer development and progression. In nontransformed cells, NF-κB activation is tightly controlled by IκBs. IκBs bind NF-κB in the cytoplasm, preventing it from translocating to the nucleus to modulate gene expression. Stimuli that activate NF-κB signaling trigger IκB degradation, enabling nuclear translocation of NF-κB. Among the genes regulated by NF-κB are those encoding the IκBs, providing a negative feedback loop that limits NF-κB activity. How transformed cells override this NF-κB/IκB negative feedback loop remains unclear. Here, we report in human glioma cell lines that microRNA-30e* (miR-30e*) directly targets the IκBα 3ι-UTR and suppresses IκBα expression. Overexpression of miR-30e* in human glioma cell lines led to hyperactivation of NF-κB and enhanced expression of NF-κB–regulated genes, which promoted glioma cell invasiveness in in vitro assays and in an orthotopic xenotransplantation model. These effects of miR-30e* were shown to be clinically relevant, as miR-30e* was found to be upregulated in primary human glioma cells and correlated with malignant progression and poor survival. Hence, miR-30e* provides an epigenetic mechanism that disrupts the NF-κB/IκBα loop and may represent a new therapeutic target and prognostic marker.
Authors
Lili Jiang, Chuyong Lin, Libing Song, Jueheng Wu, Baixue Chen, Zhe Ying, Lishan Fang, Xiao Yan, Mian He, Jun Li, Mengfeng Li
Abstract
Ectodermal dysplasia with immune deficiency (EDI) is an immunological and developmental disorder caused by alterations in the gene encoding NF-κB essential modulator (NEMO; also known as IκB kinase γ subunit [IKKγ]). Missense mutations in the gene encoding NEMO are associated with reduced signal-induced nuclear translocation of NF-κB proteins, resulting in defective expression of NF-κB target genes. Here, we report 2 unrelated male patients with EDI, both of whom have normal NEMO coding sequences, but exhibit a marked reduction in expression of full-length NEMO protein. TLR4 stimulation of APCs from these patients induced normal cytoplasmic activation and nuclear translocation of NF-κB. However, cells deficient in full-length NEMO were defective in expression of NF-κB–regulated cytokines, such as IL-12, suggesting a downstream defect in chromatin accessibility for NF-κB transcription factors. TLR4-stimulated APCs from the patients were defective in IKKα-dependent H3 histone phosphorylation at the IL-12 promoter and recruitment of NF-κB heterodimers RelA and cRel to the promoter. Expression of a super-active form of IKKα restored IL-12 production in a NEMO knockdown human monocytic cell line following LPS treatment. Our findings suggest that NEMO regulates the nuclear function of IKKα and offer new insights into the mechanisms underlying diminished NF-κB signaling in patients with EDI.
Authors
Stephane T. Temmerman, Chi A. Ma, Yongge Zhao, Jeffrey Keenan, Ivona Aksentijevich, Margaret Fessler, Margaret R. Brown, Alan Knutsen, Ralph Shapiro, Ashish Jain
Abstract
Amyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron degeneration, which ultimately leads to paralysis and death. Mutation of TAR DNA binding protein 43 (TDP-43) has been linked to the development of an inherited form of ALS. Existing TDP-43 transgenic animals develop a limited loss of motor neurons and therefore do not faithfully reproduce the core phenotype of ALS. Here, we report the creation of multiple lines of transgenic rats in which expression of ALS-associated mutant human TDP-43 is restricted to either motor neurons or other types of neurons and skeletal muscle and can be switched on and off. All of these rats developed progressive paralysis reminiscent of ALS when the transgene was switched on. Rats expressing mutant TDP-43 in motor neurons alone lost more spinal motor neurons than rats expressing the disease gene in varying neurons and muscle cells, although these rats all developed remarkable denervation atrophy of skeletal muscles. Intriguingly, progression of the disease was halted after transgene expression was switched off; in rats with limited loss of motor neurons, we observed a dramatic recovery of motor function, but in rats with profound loss of motor neurons, we only observed a moderate recovery of motor function. Our finding suggests that mutant TDP-43 in motor neurons is sufficient to promote the onset and progression of ALS and that motor neuron degeneration is partially reversible, at least in mutant TDP-43 transgenic rats.
Authors
Cao Huang, Jianbin Tong, Fangfang Bi, Hongxia Zhou, Xu-Gang Xia
Abstract
It is estimated that one-third of the world’s population is infected with Mycobacterium tuberculosis. Infection typically remains latent, but it can reactivate to cause clinical disease. The only vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), is largely ineffective, and ways to enhance its efficacy are being developed. Of note, the candidate booster vaccines currently under clinical development have been designed to improve BCG efficacy but not prevent reactivation of latent infection. Here, we demonstrate that administering a multistage vaccine that we term H56 in the adjuvant IC31 as a boost to vaccination with BCG delays and reduces clinical disease in cynomolgus macaques challenged with M. tuberculosis and prevents reactivation of latent infection. H56 contains Ag85B and ESAT-6, which are two of the M. tuberculosis antigens secreted in the acute phase of infection, and the nutrient stress–induced antigen Rv2660c. Boosting with H56/IC31 resulted in efficient containment of M. tuberculosis infection and reduced rates of clinical disease, as measured by clinical parameters, inflammatory markers, and improved survival of the animals compared with BCG alone. Boosted animals showed reduced pulmonary pathology and extrapulmonary dissemination, and protection correlated with a strong recall response against ESAT-6 and Rv2660c. Importantly, BCG/H56-vaccinated monkeys did not reactivate latent infection after treatment with anti-TNF antibody. Our results indicate that H56/IC31 boosting is able to control late-stage infection with M. tuberculosis and contain latent tuberculosis, providing a rationale for the clinical development of H56.
Authors
Philana Ling Lin, Jes Dietrich, Esterlina Tan, Rodolfo M. Abalos, Jasmin Burgos, Carolyn Bigbee, Matthew Bigbee, Leslie Milk, Hannah P. Gideon, Mark Rodgers, Catherine Cochran, Kristi M. Guinn, David R. Sherman, Edwin Klein, Christopher Janssen, JoAnne L. Flynn, Peter Andersen
Abstract
DNA methyltransferase 3B (Dnmt3b) belongs to a family of enzymes responsible for methylation of cytosine residues in mammals. DNA methylation contributes to the epigenetic control of gene transcription and is deregulated in virtually all human tumors. To better understand the generation of cancer-specific methylation patterns, we genetically inactivated Dnmt3b in a mouse model of MYC-induced lymphomagenesis. Ablation of Dnmt3b function using a conditional knockout in T cells accelerated lymphomagenesis by increasing cellular proliferation, which suggests that Dnmt3b functions as a tumor suppressor. Global methylation profiling revealed numerous gene promoters as potential targets of Dnmt3b activity, the majority of which were demethylated in Dnmt3b–/– lymphomas, but not in Dnmt3b–/– pretumor thymocytes, implicating Dnmt3b in maintenance of cytosine methylation in cancer. Functional analysis identified the gene Gm128 (which we termed herein methylated in normal thymocytes [Ment]) as a target of Dnmt3b activity. We found that Ment was gradually demethylated and overexpressed during tumor progression in Dnmt3b–/– lymphomas. Similarly, MENT was overexpressed in 67% of human lymphomas, and its transcription inversely correlated with methylation and levels of DNMT3B. Importantly, knockdown of Ment inhibited growth of mouse and human cells, whereas overexpression of Ment provided Dnmt3b+/+ cells with a proliferative advantage. Our findings identify Ment as an enhancer of lymphomagenesis that contributes to the tumor suppressor function of Dnmt3b and suggest it could be a potential target for anticancer therapies.
Authors
Ryan A. Hlady, Slavomira Novakova, Jana Opavska, David Klinkebiel, Staci L. Peters, Juraj Bies, Jay Hannah, Javeed Iqbal, Kristi M. Anderson, Hollie M. Siebler, Lynette M. Smith, Timothy C. Greiner, Dhundy Bastola, Shantaram Joshi, Oksana Lockridge, Melanie A. Simpson, Dean W. Felsher, Kay-Uwe Wagner, Wing C. Chan, Judith K. Christman, Rene Opavsky
Abstract
Mutations in the coactivator CREB-binding protein (CBP) are a major cause of the human skeletal dysplasia Rubinstein-Taybi syndrome (RTS); however, the mechanism by which these mutations affect skeletal mineralization and patterning is unknown. Here, we report the identification of 3-phosphoinositide-dependent kinase 1 (PDK1) as a key regulator of CBP activity and demonstrate that its functions map to both osteoprogenitor cells and mature osteoblasts. In osteoblasts, PDK1 activated the CREB/CBP complex, which in turn controlled runt-related transcription factor 2 (RUNX2) activation and expression of bone morphogenetic protein 2 (BMP2). These pathways also operated in vivo, as evidenced by recapitulation of RTS spectrum phenotypes with osteoblast-specific Pdk1 deletion in mice (Pdk1osx mice) and by the genetic interactions observed in mice heterozygous for both osteoblast-specific Pdk1 deletion and either Runx2 or Creb deletion. Finally, treatment of Pdk1osx and Cbp+/– embryos with BMPs in utero partially reversed their skeletal anomalies at birth. These findings illustrate the in vivo function of the PDK1-AKT-CREB/CBP pathway in bone formation and provide proof of principle for in utero growth factor supplementation as a potential therapy for skeletal dysplasias.
Authors
Jae-Hyuck Shim, Matthew B. Greenblatt, Anju Singh, Nicholas Brady, Dorothy Hu, Rebecca Drapp, Wataru Ogawa, Masato Kasuga, Tetsuo Noda, Sang-Hwa Yang, Sang-Kyou Lee, Vivienne I. Rebel, Laurie H. Glimcher
Abstract
Oncogenic Ras and p53 loss-of-function mutations are common in many advanced sporadic malignancies and together predict a limited responsiveness to conventional chemotherapy. Notably, studies in cultured cells have indicated that each of these genetic alterations creates a selective sensitivity to ataxia telangiectasia and Rad3-related (ATR) pathway inhibition. Here, we describe a genetic system to conditionally reduce ATR expression to 10% of normal levels in adult mice to compare the impact of this suppression on normal tissues and cancers in vivo. Hypomorphic suppression of ATR minimally affected normal bone marrow and intestinal homeostasis, indicating that this level of ATR expression was sufficient for highly proliferative adult tissues. In contrast, hypomorphic ATR reduction potently inhibited the growth of both p53-deficient fibrosarcomas expressing H-rasG12V and acute myeloid leukemias (AMLs) driven by MLL-ENL and N-rasG12D. Notably, DNA damage increased in a greater-than-additive fashion upon combining ATR suppression with oncogenic stress (H-rasG12V, K-rasG12D, or c-Myc overexpression), indicating that this cooperative genome-destabilizing interaction may contribute to tumor selectivity in vivo. This toxic interaction between ATR suppression and oncogenic stress occurred without regard to p53 status. These studies define a level of ATR pathway inhibition in which the growth of malignancies harboring oncogenic mutations can be suppressed with minimal impact on normal tissue homeostasis, highlighting ATR inhibition as a promising therapeutic strategy.
Authors
David W. Schoppy, Ryan L. Ragland, Oren Gilad, Nishita Shastri, Ashley A. Peters, Matilde Murga, Oscar Fernandez-Capetillo, J. Alan Diehl, Eric J. Brown
Abstract
Type 1 diabetes (T1D) is caused by autoimmune destruction of the insulin-producing β cells in the pancreatic islets, which are essentially mini-organs embedded in exocrine tissue. CTLs are considered to have a predominant role in the autoimmune destruction underlying T1D. Visualization of CTL-mediated killing of β cells would provide new insight into the pathogenesis of T1D, but has been technically challenging to achieve. Here, we report our use of intravital 2-photon imaging in mice to visualize the dynamic behavior of a virally expanded, diabetogenic CTL population in the pancreas at cellular resolution. Following vascular arrest and extravasation, CTLs adopted a random motility pattern throughout the compact exocrine tissue and displayed unimpeded yet nonlinear migration between anatomically nearby islets. Upon antigen encounter within islets, a confined motility pattern was acquired that allowed the CTLs to scan the target cell surface. A minority of infiltrating CTLs subsequently arrested at the β cell junction, while duration of stable CTL–target cell contact was on the order of hours. Slow-rate killing occurred in the sustained local presence of substantial numbers of effector cells. Collectively, these data portray the kinetics of CTL homing to and between antigenic target sites as a stochastic process at the sub-organ level and argue against a dominant influence of chemotactic gradients.
Authors
Ken Coppieters, Natalie Amirian, Matthias von Herrath
Abstract
Antagonists of L-type Ca2+ channels (LTCCs) have been used to treat human cardiovascular diseases for decades. However, these inhibitors can have untoward effects in patients with heart failure, and their overall therapeutic profile remains nebulous given differential effects in the vasculature when compared with those in cardiomyocytes. To investigate this issue, we examined mice heterozygous for the gene encoding the pore-forming subunit of LTCC (calcium channel, voltage-dependent, L type, α1C subunit [Cacna1c mice; referred to herein as α1C–/+ mice]) and mice in which this gene was loxP targeted to achieve graded heart-specific gene deletion (termed herein α1C-loxP mice). Adult cardiomyocytes from the hearts of α1C–/+ mice at 10 weeks of age showed a decrease in LTCC current and a modest decrease in cardiac function, which we initially hypothesized would be cardioprotective. However, α1C–/+ mice subjected to pressure overload stimulation, isoproterenol infusion, and swimming showed greater cardiac hypertrophy, greater reductions in ventricular performance, and greater ventricular dilation than α1C+/+ controls. The same detrimental effects were observed in α1C-loxP animals with a cardiomyocyte-specific deletion of one allele. More severe reductions in α1C protein levels with combinatorial deleted alleles produced spontaneous cardiac hypertrophy before 3 months of age, with early adulthood lethality. Mechanistically, our data suggest that a reduction in LTCC current leads to neuroendocrine stress, with sensitized and leaky sarcoplasmic reticulum Ca2+ release as a compensatory mechanism to preserve contractility. This state results in calcineurin/nuclear factor of activated T cells signaling that promotes hypertrophy and disease.
Authors
Sanjeewa A. Goonasekera, Karin Hammer, Mannix Auger-Messier, Ilona Bodi, Xiongwen Chen, Hongyu Zhang, Steven Reiken, John W. Elrod, Robert N. Correll, Allen J. York, Michelle A. Sargent, Franz Hofmann, Sven Moosmang, Andrew R. Marks, Steven R. Houser, Donald M. Bers, Jeffery D. Molkentin
Abstract
Spinal cord injury (SCI) often leads to persistent functional deficits due to loss of neurons and glia and to limited axonal regeneration after injury. Here we report that transplantation of human dental pulp stem cells into the completely transected adult rat spinal cord resulted in marked recovery of hind limb locomotor functions. Transplantation of human bone marrow stromal cells or skin-derived fibroblasts led to substantially less recovery of locomotor function. The human dental pulp stem cells exhibited three major neuroregenerative activities. First, they inhibited the SCI-induced apoptosis of neurons, astrocytes, and oligodendrocytes, which improved the preservation of neuronal filaments and myelin sheaths. Second, they promoted the regeneration of transected axons by directly inhibiting multiple axon growth inhibitors, including chondroitin sulfate proteoglycan and myelin-associated glycoprotein, via paracrine mechanisms. Last, they replaced lost cells by differentiating into mature oligodendrocytes under the extreme conditions of SCI. Our data demonstrate that tooth-derived stem cells may provide therapeutic benefits for treating SCI through both cell-autonomous and paracrine neuroregenerative activities.
Authors
Kiyoshi Sakai, Akihito Yamamoto, Kohki Matsubara, Shoko Nakamura, Mami Naruse, Mari Yamagata, Kazuma Sakamoto, Ryoji Tauchi, Norimitsu Wakao, Shiro Imagama, Hideharu Hibi, Kenji Kadomatsu, Naoki Ishiguro, Minoru Ueda
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ISSN: 0021-9738 (print), 1558-8238 (online)