Shigeki Miyamoto

University of Wisconsin–Madison

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Co-Authors

Natalie S. Callander

University of Wisconsin–Madison

Peiman Hematti

Medical College of Wisconsin

Nozomu Sasaki

Saitama Medical University

Shelly M. Wuerzberger‐Davis

University of Wisconsin–Madison

Fotis Asimakopoulos

University of California, San Diego

Chelsea Hope

Lutheran Family Health Centers

Hideaki Karaki

The University of Tokyo

Hiroo Niimi

Chiba University

Hirokazu Sato

Hokkaido Research Organization

Stephanie Markovina

Washington University in St. Louis

Cooperative Institutions

Inserm

270

Centre National de la Recherche Scientifique

228

Harvard University

219

University of Wisconsin–Madison

216

University of Michigan–Ann Arbor

198

National Institutes of Health

160

Chinese Academy of Sciences

151

University of California, San Diego

126

Istituti di Ricovero e Cura a Carattere Scientifico

123

Johns Hopkins University

113

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CRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold protein

PLoS ONE (2019)

Milad Babaei Yuekun Liu Shelly M. Wuerzberger‐Davis Ethan Z. McCaslin Christopher J. DiRusso

The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-κB signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter D) is largely responsible for NEMO transcription in liver cells. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support activation of canonical NF-κB signaling in response to treatment with tumor necrosis factor. Nevertheless, non-canonical NF-κB signaling is intact in these NEMO-deficient cells. Expression of ectopic wild-type NEMO, but not certain human NEMO disease mutants, in the edited cells restores downstream NF-κB signaling in response to tumor necrosis factor. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter D) in the human SNU-423 liver cancer cell line. Thus, we have created a strategy for selectively eliminating cell type-specific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-κB signaling are discussed.

HEK 293 cells

Cell type

10.1371/journal.pone.0222588

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Citations (11)

HAPLN1 matrikine: a bone marrow homing factor linked to poor outcomes in patients with MM

Blood Advances (2023)

Hae Yeun Chang Mailee Huynh Avtar Roopra Natalie S. Callander Shigeki Miyamoto

The bone marrow (BM) microenvironment is critical for dissemination, growth, and survival of multiple myeloma (MM) cells. Homing of myeloma cells to the BM niche is a crucial step in MM dissemination, but the mechanisms involved are incompletely understood. In particular, any role of matrikines, neofunctional peptides derived from extracellular matrix proteins, remains unknown. Here, we report that a matrikine derived from hyaluronan and proteoglycan link protein 1 (HAPLN1) induces MM cell adhesion to the BM stromal components, such as fibronectin, endothelial cells, and stromal cells and, furthermore, induces their chemotactic and chemokinetic migration. In a mouse xenograft model, we show that MM cells preferentially home to HAPLN1 matrikine-conditioned BM. The transcription factor STAT1 is activated by HAPLN1 matrikine and is necessary to induce MM cell adhesion, migration, migration-related genes, and BM homing. STAT1 activation is mediated by interferon beta (IFN-β), which is induced by NF-κB after stimulation by HAPLN1 matrikine. Finally, we also provide evidence that higher levels of HAPLN1 in BM samples correlate with poorer progression-free survival of patients with newly diagnosed MM. These data reveal that a matrikine present in the BM microenvironment acts as a chemoattractant, plays an important role in BM homing of MM cells via NF-κB-IFN-β-STAT1 signaling, and may help identify patients with poor outcomes. This study also provides a mechanistic rationale for targeting HAPLN1 matrikine in MM therapy.

Homing (biology)

10.1182/bloodadvances.2023010139

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Citations (3)

Oita International Electrocardiology Symposium 2000 “Electrophysiology and Management of Lethal Arrhythmias in the New Millennium: From Genes to Bedside”

Japanese Journal of Electrocardiology (2000)

Keitaro Hashimoto Atsushi Sugiyama Shigeki Miyamoto Yi Xue Nu Nu Aye

Cariporide and KB-R9032, new Na-H exchange (NHE) subtype 1 inhibitors, have antiarrhythmic effects on canine coronary ischemia and reperfusion induced arrhythmias, which might be induced by intracellular Ca overload following myocardial ischemia. These favorable antiarrhythmic effects on coronary ischemia and reperfusion occurred without changes in the blood pressure, heart rate and ECG parameters. Such antiarrhythmic and antifibrillatory effects were specific for myocardial ischemia related arrhythmias, namely they did not show such antiarrhythmic effects on digitalis or adrenaline induced arrhythmias, the mechanisms of which are also thought to be due to intracellular Ca overload. A new selective Na/Ca exchange inhibitor, KBR7943, was proved to be a reverse mode inhibitor, but failed to show antiarrhythmic effects on the canine coronary ischemia and reperfusion induced arrhythmias.

10.5105/jse.20.suppl3_101

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Citations (2)

Species- and Temperature-Dependency of the Decrease in Myofilament Ca2+ Sensitivity Induced by β -Adrenergic Stimulation

The Japanese Journal of Pharmacology (2001)

Shigeki Miyamoto Masatoshi Hori Masanori Izumi Hiroshi Ozaki Hideaki Karaki

Although beta-adrenergic stimulation has been shown in many studies to decrease myofilament Ca2+ sensitivity in various types of cardiac muscle such as rat and rabbit ventricles, other studies disagree with this conclusion. In the present study, we aimed to explain these contradictory findings. We examined the effect of beta-adrenoceptor stimulation on Ca2+ sensitivity using guinea pig and rat ventricles. We performed the experiment at two different temperatures and compared the results. In guinea pig ventricles, isoproterenol and forskolin did not alter the relationship between [Ca2+]i and muscle force during the relaxation phase of tetanic contraction at either 24 degrees C or 30 degrees C. In rat ventricles, in contrast, isoproterenol shifted the [Ca2+]i-force curve to the right at 24 degrees C, but not at 30 degrees C. In guinea pig ventricles permeabilized by alpha-toxin, in which the cAMP/PK-A system is intact, the addition of cAMP did not decrease Ca2+ sensitivity. These results suggest that there are species- and temperature-dependent differences in the regulation of myofilament Ca2+ sensitivity by beta-adrenergic stimulation.

Myofilament

Isoprenaline

10.1254/jjp.85.75

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Citations (6)

A Cytosolic ATM/NEMO/RIP1 Complex Recruits TAK1 To Mediate the NF-κB and p38 Mitogen-Activated Protein Kinase (MAPK)/MAPK-Activated Protein 2 Responses to DNA Damage

Molecular and Cellular Biology (2011)

Yibin Yang Fang Xia Nicole Hermance Angela M. Mabb Sara J.S. Simonson

AbstractIn multiple tumor types, activation of the transcription factor NF-κB increases the resistance of tumor cells to anticancer therapies and contributes to tumor progression. Genotoxic stress induced by chemotherapy or radiation therapy triggers the ATM-dependent translocation of NF-κB essential modifier (NEMO), also designated IκB kinase γ (IKKγ), from the nucleus to the cytosol, resulting in IκB kinase activation by mechanisms not yet fully understood. RIP1 has been implicated in this response and found to be modified in cells with damaged DNA; however, the nature of the RIP1 modification and its precise role in the pathway remain unclear. Here, we show that DNA damage stimulates the formation of a cytosolic complex containing ATM, NEMO (IKKγ), RIP1, and TAK1. We find that RIP1 is modified by SUMO-1 and ubiquitin in response to DNA damage and demonstrate that modified RIP1 is required for NF-κB activation and tumor cell survival. We show that ATM activates TAK1 in a manner dependent on RIP1 and NEMO. We also reveal TAK1 as a central mediator of the alternative DNA damage response pathway mediated by the p38 mitogen-activated protein kinase (MAPK)/MAPK-activated protein 2 (MAPKAP-2) kinases. These findings have translational implications and reveal RIP1 and TAK1 as potential therapeutic targets in chemoresistance. ACKNOWLEDGMENTSWe thank Sankar Ghosh for the control and Tak1−/− MEFs, Manolis Pasparakis for the control and Nemo−/− MEFs, and Stephen Jones for the control and Atm−/− MEFs used in this study. We thank Zhijian (James) Chen for providing the His-tagged, kinase-inactive MKK6(K82A) substrate. We thank Neal Silverman for critical reading of the manuscript.We also gratefully acknowledge that Core resources supported by Diabetes Endocrinology Research Center grant DK32520 were also used. This work was supported by grant RO1 AI075118 to M.A.K. and grants RO1 CA77474 and GM083681 to S.M.

IκB kinase

10.1128/mcb.01139-10

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Citations (122)

NF-κB Activation by Camptothecin

Journal of Biological Chemistry (2000)

Tony T. Huang Shelly M. Wuerzberger‐Davis Bradley J. Seufzer Stuart D. Shumway Takeshi Kurama

Activation of the transcription factor NF-κB by extracellular signals involves its release from the inhibitor protein IκBα in the cytoplasm and subsequent nuclear translocation. NF-κB can also be activated by the anticancer agent camptothecin (CPT), which inhibits DNA topoisomerase (Topo) I activity and causes DNA double-strand breaks during DNA replication to induce S phase-dependent cytotoxicity. Here we show that CPT activates NF-κB by a mechanism that is dependent on initial nuclear DNA damage followed by cytoplasmic signaling events. NF-κB activation by CPT is dramatically diminished in cytoplasts and in CEM/C2 cells expressing a mutant Topo I protein that fails to bind CPT. This response is intensified in S phase cell populations and is prevented by the DNA polymerase inhibitor aphidicolin. In addition, CPT activation of NF-κB involves degradation of cytoplasmic IκBα by the ubiquitin-proteasome pathway in a manner that depends on the IκB kinase complex. Finally, inhibition of NF-κB activation augments CPT-induced apoptosis. These findings elucidate the progression of signaling events that initiates in the nucleus with CPT-Topo I interaction and continues in the cytoplasm resulting in degradation of IκBα and nuclear translocation of NF-κB to attenuate the apoptotic response.

Camptothecin

10.1074/jbc.275.13.9501

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Citations (153)

Data from Bortezomib-Resistant Nuclear Factor-κB Activity in Multiple Myeloma Cells

Stephanie Markovina Natalie S. Callander Shelby L. O’Connor Jihoon Kim Jae Werndli

<div>Abstract<p>Bortezomib (Velcade/PS341), a proteasome inhibitor used in the treatment of multiple myeloma (MM), can inhibit activation of nuclear factor-κB (NF-κB), a family of transcription factors often deregulated and constitutively activated in primary MM cells. NF-κB can be activated via several distinct mechanisms, including the proteasome inhibitor–resistant (PIR) pathway. It remains unknown what fraction of primary MM cells harbor constitutive NF-κB activity maintained by proteasome-dependent mechanisms. Here, we report an unexpected finding that constitutive NF-κB activity in 10 of 14 primary MM samples analyzed is refractory to inhibition by bortezomib. Moreover, when MM cells were cocultured with MM patient-derived bone marrow stromal cells (BMSC), microenvironment components critical for MM growth and survival, further increases in NF-κB activity were observed that were also refractory to bortezomib. Similarly, MM-BMSCs caused PIR NF-κB activation in the RPMI8226 MM cell line, leading to increased NF-κB–dependent transcription and resistance to bortezomib-induced apoptosis. Our findings show that primary MM cells frequently harbor PIR NF-κB activity that is further enhanced by the presence of patient-derived BMSCs. They also suggest that this activity is likely relevant to the drug resistance development in some patients. Further elucidation of the mechanism of PIR NF-κB regulation could lead to the identification of novel diagnostic biomarkers and/or therapeutic targets for MM treatment. (Mol Cancer Res 2008;6(8):1356–64)</p></div>

IκBα

10.1158/1541-7786.c.6542187

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Citations (0)

Inhibition of IκBα Nuclear Export as an Approach to Abrogate Nuclear Factor-κB–Dependent Cancer Cell Survival

Molecular Cancer Research (2005)

Shelby L. O’Connor Stuart D. Shumway Shigeki Miyamoto

Abstract Deregulation of the transcription factor nuclear factor-κB (NF-κB) leading to its constitutive activation is frequently observed in human cancer. Because altered NF-κB activities often promote the survival of malignant cells, its inhibition is regarded as a promising anticancer strategy. Because activation of the latent cytoplasmic NF-κB complex can be induced by a wide variety of different stimuli, its deregulation may occur by an equally large number of distinct mechanisms. This diversity raises a conundrum in conceptualizing general approaches to attenuate NF-κB activity in cancer. Here, we provide evidence that inhibition of IκBα nuclear export is a viable target to generally abrogate constitutive NF-κB activity in different cancer cell types. We show that inhibition of IκBα nuclear export has an important course of events in cancer cells harboring constitutive NF-κB activity—an initial increase in the pool of stable nuclear NF-κB/IκBα complexes that leads to a reduction of constitutive NF-κB activity and subsequent induction of apoptosis. Importantly, similar effects on multiple different cancer cell types indicate that inhibition of nuclear export of IκBα leads to broad inhibition of constitutive NF-κB activation regardless of various deregulated, upstream events involved.

Nuclear export signal

IκB kinase

IκBα

10.1158/1541-7786.42.3.1

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Citations (10)

Identification of two novel amino acid polymorphisms in beta-cell/liver (GLUT2) glucose transporter in Japanese subjects

Diabetologia (1995)

F Shimada Hideichi Makino Hiroko Iwaoka Shigeki Miyamoto Naotake Hashimoto

GLUT2

Single-strand conformation polymorphism

10.1007/bf00400096