Publications by authors named "Ja Hyun Koo"

41 Publications

Ablation of USP21 in skeletal muscle promotes oxidative fibre phenotype, inhibiting obesity and type 2 diabetes.

J Cachexia Sarcopenia Muscle 2021 Sep 15. Epub 2021 Sep 15.

College of Pharmacy and Integrated Research Institute for Drug Development, Dongguk University-Seoul, Goyang-si, Gyeonggi-Do, South Korea.

Background: Skeletal muscle as a metabolic consumer determines systemic energy homeostasis by regulating myofibre type conversion and muscle mass control. Perturbation of the skeletal muscle metabolism elevates the risk of a variety of diseases including metabolic disorders. However, the regulatory pathways and molecules are not completely understood. The discovery of relevant responsible molecules and the associated network could be an attractive strategy to overcome diseases associated with muscle problems.

Methods: An initial screening using quantitative trait locus analysis enabled us to extract a set of genes including ubiquitin-specific proteases21 (USP21) (r = 0.738; P = 0.004) as potential targets associated with fasting blood glucose content. Given tight regulation of the ubiquitination status of proteins in muscle, we focused on USP21 and generated whole-body (KO) and skeletal muscle-specific USP21 knockout (MKO) mice. Transcriptomics, proteomics, and lipidomics assays in combination with various in vivo and in vitro experiments were performed to understand the functions of USP21 and underlying mechanisms. A high-fat diet (60%)-fed mouse model and diabetic patient-derived samples were utilized to assess the effects of USP21 on energy metabolism in skeletal muscle.

Results: USP21 was highly expressed in both human and mouse skeletal muscle, and controlled skeletal muscle oxidative capacity and fuel consumption. USP21-KO or USP21-MKO significantly promoted oxidative fibre type changes (Δ36.6% or Δ47.2%), muscle mass increase (Δ13.8% to Δ22.8%), and energy expenditure through mitochondrial biogenesis, fatty acid oxidation, and UCP2/3 induction (P < 0.05 or P < 0.01). Consistently, cold exposure repressed USP21 expression in mouse skeletal muscle (Δ55.3%), whereas loss of USP21 increased thermogenesis (+1.37°C or +0.84°C; P < 0.01). Mechanistically, USP21 deubiquitinated DNA-PKcs and ACLY, which led to AMPK inhibition. Consequently, USP21 ablation diminished diet-induced obesity (WT vs. USP21-KO, Δ8.02 g, 17.1%, P < 0.01; litter vs. USP21-MKO, Δ3.48 g, 7.7%, P < 0.05) and insulin resistance. These findings were corroborated in a skeletal muscle-specific gene KO mouse model. USP21 was induced in skeletal muscle of a diabetic patient (1.94-fold), which was reciprocally changed to p-AMPK (0.30-fold).

Conclusions: The outcomes of this research provide novel information as to how USP21 in skeletal muscle contributes to systemic energy homeostasis, demonstrating USP21 as a key molecule in the regulation of myofibre type switch, muscle mass control, mitochondrial function, and heat generation and, thus, implicating the potential of this molecule and its downstream substrates network as targets for the treatment and/or prevention of muscle dysfunction and the associated metabolic diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jcsm.12777DOI Listing
September 2021

In Vivo Induction of Regulatory T Cells Via CTLA-4 Signaling Peptide to Control Autoimmune Encephalomyelitis and Prevent Disease Relapse.

Adv Sci (Weinh) 2021 07 5;8(14):2004973. Epub 2021 May 5.

Department of Life Science College of Natural Sciences Hanyang University Research institute for Natural Sciences Research Institute for Convergence of Basic Sciences Hanyang University Seoul 04763 Republic of Korea.

Regulatory T cells play a key role in immune tolerance to self-antigens, thereby preventing autoimmune diseases. However, no drugs targeting Treg cells have been approved for clinical trials yet. Here, a chimeric peptide is generated by conjugation of the cytoplasmic domain of CTLA-4 (ctCTLA-4) with dNP2 for intracellular delivery, dNP2-ctCTLA-4, and evaluated Foxp3 expression during Th0, Th1, Treg, and Th17 differentiation dependent on TGF-. The lysine motif of ctCTLA-4, not tyrosine motif, is required for Foxp3 expression for Treg induction and amelioration of experimental autoimmune encephalomyelitis (EAE). Transcriptome analysis reveals that dNP2-ctCTLA-4-treated T cells express Treg transcriptomic patterns with properties of suppressive functions. In addition, the molecular interaction between the lysine motif of ctCTLA-4 and PKC- is critical for Foxp3 expression. Although both CTLA-4-Ig and dNP2-ctCTLA-4 treatment in vivo ameliorated EAE progression, only dNP2-ctCTLA-4 requires Treg cells for inhibition of disease progression and prevention of relapse. Furthermore, the CTLA-4 signaling peptide is able to induce human Tregs in vitro and in vivo as well as from peripheral blood mononuclear cells (PBMCs) of multiple sclerosis patients. These results collectively suggest that the chimeric CTLA-4 signaling peptide can be used for successful induction of regulatory T cells in vivo to control autoimmune diseases, such as multiple sclerosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/advs.202004973DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8292875PMC
July 2021

Dickkopf-2 regulates the stem cell marker LGR5 in colorectal cancer via HNF4α1.

iScience 2021 May 15;24(5):102411. Epub 2021 Apr 15.

Department of Immunobiology, Yale University School of Medicine, TAC 641D, PO Box 208011, 300 Cedar Street, New Haven, CT 06520-8011, USA.

Enhanced stemness in colorectal cancer has been reported and it contributes to aggressive progression, but the underlying mechanisms remain unclear. Here we report a Wnt ligand, Dickkopf-2 (DKK2) is essential for developing colorectal cancer stemness. Genetic depletion of in intestinal epithelial or stem cells reduced tumorigenesis and expression of the stem cell marker genes including in a model of colitis-associated cancer. Sequential mutations in , , , and genes in colonic organoids revealed a significant increase of expression by APC knockout and further increased by additional and mutations. Moreover, DKK2 activates proto-oncogene tyrosine-protein kinse Src followed by increased LGR5 expressing cells in colorectal cancer through degradation of HNF4α1 protein. These findings suggest that DKK2 is required for colonic epithelial cells to enhance LGR5 expression during the progression of colorectal cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.isci.2021.102411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099562PMC
May 2021

Macrophage-preferable delivery of the leucine-rich repeat domain of NLRX1 ameliorates lethal sepsis by regulating NF-κB and inflammasome signaling activation.

Biomaterials 2021 07 4;274:120845. Epub 2021 May 4.

Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, 04763, Republic of Korea; Research Institute for Natural Sciences, Hanyang University, Seoul, 04763, Republic of Korea; Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul, 04763, Republic of Korea. Electronic address:

Sepsis is an acute systemic inflammatory disease triggered by bacterial infection leading organ dysfunctions that macrophages are responsible for major triggering of systemic inflammation. Treatment options are limited to antibiotics and drugs to manage the symptoms of sepsis, but there are currently no molecular-targeted therapies. Here, we identified a novel macrophage-preferable delivery peptide, C10, which we conjugated to truncated domains of NLRX1 (leucine-rich repeat region (LRR), and nucleotide binding domain (NBD)) to obtain C10-LRR and C10-NBD. Leucine rich amino acid of C10 enables macrophage preferable moieties that efficiently deliver a cargo protein into macrophages in vitro and in vivo. C10-LRR but not C10-NBD significantly improved survival in an LPS-mediated lethal endotoxemia sepsis model. C10-LRR efficiently inhibited IL-6 production in peritoneal macrophages via prevention of IκB degradation and p65 phosphorylation. In addition, C10-LRR negatively regulated IL-1β production by preventing caspase-1 activation with a sustained mitochondrial MAVS level. Finally, co-treatment with anti-TNFα antibody and C10-LRR had a synergistic effect in an LPS-induced sepsis model. Collectively, these findings indicate that C10-LRR could be an effective therapeutic agent to treat systemic inflammation in sepsis by regulating both NF-κB and inflammasome signaling activation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biomaterials.2021.120845DOI Listing
July 2021

Signaling Nodes Associated with Endoplasmic Reticulum Stress during NAFLD Progression.

Biomolecules 2021 02 8;11(2). Epub 2021 Feb 8.

School of Pharmacy and Institute of New Drug Development, Jeonbuk National University, 567 Baekje-daero, Deokjin-gu, Jeonju-si, Jeollabuk-do 54896, Korea.

Excess and sustained endoplasmic reticulum (ER) stress, paired with a failure of initial adaptive responses, acts as a critical trigger of nonalcoholic fatty liver disease (NAFLD) progression. Unfortunately, there is no drug currently approved for treatment, and the molecular basis of pathogenesis by ER stress remains poorly understood. Classical ER stress pathway molecules have distinct but inter-connected functions and complicated effects at each phase of the disease. Identification of the specific molecular signal mediators of the ER stress-mediated pathogenesis is, therefore, a crucial step in the development of new treatments. These signaling nodes may be specific to the cell type and/or the phase of disease progression. In this review, we highlight the recent advancements in knowledge concerning signaling nodes associated with ER stress and NAFLD progression in various types of liver cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/biom11020242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7915814PMC
February 2021

T-Cell-Mimicking Nanoparticles for Cancer Immunotherapy.

Adv Mater 2020 Oct 18;32(39):e2003368. Epub 2020 Aug 18.

Interdisciplinary Program for Bioengineering, Seoul National University, Seoul, 08826, Republic of Korea.

Cancer immunotherapies, including adoptive T cell transfer and immune checkpoint blockades, have recently shown considerable success in cancer treatment. Nevertheless, transferred T cells often become exhausted because of the immunosuppressive tumor microenvironment. Immune checkpoint blockades, in contrast, can reinvigorate the exhausted T cells; however, the therapeutic efficacy is modest in 70-80% of patients. To address some of the challenges faced by the current cancer treatments, here T-cell-membrane-coated nanoparticles (TCMNPs) are developed for cancer immunotherapy. Similar to cytotoxic T cells, TCMNPs can be targeted at tumors via T-cell-membrane-originated proteins and kill cancer cells by releasing anticancer molecules and inducing Fas-ligand-mediated apoptosis. Unlike cytotoxic T cells, TCMNPs are resistant to immunosuppressive molecules (e.g., transforming growth factor-β1 (TGF-β1)) and programmed death-ligand 1 (PD-L1) of cancer cells by scavenging TGF-β1 and PD-L1. Indeed, TCMNPs exhibit higher therapeutic efficacy than an immune checkpoint blockade in melanoma treatment. Furthermore, the anti-tumoral actions of TCMNPs are also demonstrated in the treatment of lung cancer in an antigen-nonspecific manner. Taken together, TCMNPs have a potential to improve the current cancer immunotherapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/adma.202003368DOI Listing
October 2020

LRR domain of NLRX1 protein delivery by dNP2 inhibits T cell functions and alleviates autoimmune encephalomyelitis.

Theranostics 2020 10;10(7):3138-3150. Epub 2020 Feb 10.

Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 04763, Republic of Korea.

Multiple sclerosis (MS) is a demyelinating inflammatory disease of the central nervous system (CNS), which is a chronic progressive disease and is caused by uncontrolled activation of myelin antigen specific T cells. It has high unmet medical needs due to the difficulty of efficient drug delivery into the CNS to control tissue inflammation. In this study, we demonstrate that a fusion protein of NOD-like receptor family member X1 (NLRX1) and blood brain barrier (BBB)-permeable peptide, dNP2 ameliorates experimental autoimmune encephalomyelitis (EAE). : We purified recombinant LRR or NBD regions of NLRX1 protein conjugated with dNP2. To examine intracellular delivery efficiency of the recombinant protein, we incubated the proteins with Jurkat T cells or murine splenic T cells and their delivery efficiency was analyzed by flow cytometry. To investigate the therapeutic efficacy in an EAE model, we injected the recombinant protein into mice with 3 different treatment schemes e.g., prevention, semi-therapeutic, and therapeutic. To analyze their functional roles in T cells, we treated MACS-sorted naïve CD4 T cells with the proteins during their activation and differentiation into Th1, Th17, and Treg cells. : dNP2-LRR protein treatment showed significantly higher delivery efficiency than TAT-LRR or LRR alone in Jurkat T cells and mouse splenic T cells. In all three treatment schemes of EAE experiments, dNP2-LRR administration showed ameliorated tissue inflammation and disease severity with reduced number of infiltrating T cells producing inflammatory cytokines such as IFNγ. In addition, dNP2-LRR inhibited T cell activation, cytokine production, and Th1 differentiation. : These results suggest that dNP2-LRR is a novel agent, which regulates effector T cell functions and could be a promising molecule for the treatment of CNS autoimmune diseases such as multiple sclerosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7150/thno.43441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053182PMC
August 2021

Induction of AP-1 by YAP/TAZ contributes to cell proliferation and organ growth.

Genes Dev 2020 01 12;34(1-2):72-86. Epub 2019 Dec 12.

Department of Pharmacology, Moores Cancer Center, University of California at San Diego, La Jolla, California 92093, USA.

Yes-associated protein (YAP) and its homolog transcriptional coactivator with PDZ-binding motif (TAZ) are key effectors of the Hippo pathway to control cell growth and organ size, of which dysregulation yields to tumorigenesis or hypertrophy. Upon activation, YAP/TAZ translocate into the nucleus and bind to TEAD transcription factors to promote transcriptional programs for proliferation or cell specification. Immediate early genes, represented by AP-1 complex, are rapidly induced and control later-phase transcriptional program to play key roles in tumorigenesis and organ maintenance. Here, we report that YAP/TAZ directly promote transcription that in turn contributes to the biological function of YAP/TAZ. YAP/TAZ bind to the promoter region of to stimulate its transcription. Deletion of YAP/TAZ blocks the induction of immediate early genes in response to mitogenic stimuli. induction contributes to expression of YAP/TAZ downstream target genes. Genetic deletion or chemical inhibition of AP-1 suppresses growth of YAP-driven cancer cells, such as -deficient cancer cells as well as Gα mutated uveal melanoma. Furthermore, AP-1 inhibition almost completely abrogates the hepatomegaly induced by YAP overexpression. Our findings reveal a feed-forward interplay between immediate early transcription of AP-1 and Hippo pathway function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.331546.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6938666PMC
January 2020

Overproduction of inter-α-trypsin inhibitor heavy chain 1 after loss of Gα in liver exacerbates systemic insulin resistance in mice.

Sci Transl Med 2019 10;11(513)

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 08826, Korea.

The impact of liver disease on whole-body glucose homeostasis is largely attributed to dysregulated release of secretory proteins in response to metabolic stress. The molecular cues linking liver to whole-body glucose metabolism remain elusive. We found that expression of G protein α-13 (Gα) was decreased in the liver of mice and humans with diabetes. Liver-specific deletion of the gene in mice resulted in systemic glucose intolerance. Comparative secretome analysis identified inter-α-trypsin inhibitor heavy chain 1 (ITIH1) as a protein secreted by liver that was responsible for systemic insulin resistance in deficient mice. Liver expression of ITIH1 positively correlated with surrogate markers for diabetes in patients with impaired glucose tolerance or overt diabetes. Mechanistically, a decrease in hepatic Gα caused ITIH1 oversecretion by liver through induction of -GlcNAc transferase expression, facilitating ITIH1 deposition on the hyaluronan surrounding mouse adipose tissue and skeletal muscle. Neutralization of secreted ITIH1 ameliorated glucose intolerance in obese mice. Our findings demonstrate systemic insulin resistance in mice resulting from liver-secreted ITIH1 downstream of Gα and its reversal by ITIH1 neutralization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scitranslmed.aan4735DOI Listing
October 2019

Liver X Receptor α-Induced Cannabinoid Receptor 2 Inhibits Ubiquitin-Specific Peptidase 4 Through miR-27b, Protecting Hepatocytes From TGF-β.

Hepatol Commun 2019 Oct 5;3(10):1373-1387. Epub 2019 Aug 5.

College of Pharmacy and Research Institute of Pharmaceutical Sciences Seoul National University Seoul Korea.

Liver X receptor-alpha (LXRα) acts as a double-edged sword in different biological situations. Given the elusive role of LXRα in hepatocyte viability, this study investigated whether LXRα protects hepatocytes from injurious stimuli and the underlying basis. LXRα activation prevented hepatocyte apoptosis from CCl challenges in mice. Consistently, LXRα protected hepatocytes specifically from transforming growth factor-beta (TGF-β), whereas LXRα deficiency aggravated TGF-β-induced hepatocyte injury. In the Gene Expression Omnibus database analysis for LXR mice, TGF-β receptors were placed in the core network. Hierarchical clustering and correlation analyses enabled us to find cannabinoid receptor 2 (CB2) as a gene relevant to LXRα. In human fibrotic liver samples, both LXRα and CB2 were lower in patients with septal fibrosis and cirrhosis than those with portal fibrosis. LXRα transcriptionally induced CB2; CB2 then defended hepatocytes from TGF-β. In a macrophage depletion model, JWH133 (a CB2 agonist) treatment prevented toxicant-induced liver injury. MicroRNA 27b (miR-27b) was identified as an inhibitor of ubiquitin-specific peptidase 4 (USP4), deubiquitylating TGF-β receptor 1 (TβRI), downstream from CB2. Liver-specific overexpression of LXRα protected hepatocytes from injurious stimuli and attenuated hepatic inflammation and fibrosis. : LXRα exerts a cytoprotective effect against TGF-β by transcriptionally regulating the CB2 gene in hepatocytes, and CB2 then inhibits USP4-stabilizing TβRI through miR-27b. Our data provide targets for the treatment of acute liver injury.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hep4.1415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6771303PMC
October 2019

Nrf2-lncRNA controls cell fate by modulating p53-dependent Nrf2 activation as an miRNA sponge for Plk2 and p21.

FASEB J 2019 07 21;33(7):7953-7969. Epub 2019 Mar 21.

College of Pharmacy, Seoul National University, Seoul, South Korea.

Long noncoding RNA (lncRNA) capable of controlling antioxidative capacity remains to be investigated. Nuclear factor erythroid-2-related factor 2 (Nrf2) is a central molecule for cellular defense that increases antioxidative capacity. We identified a novel lncRNA named Nrf2-activating lncRNA () transcribed from an upstream region of the microRNA 122 gene (). existed in the cytoplasm, suggestive of its function as a competing endogenous RNA [ceRNA, microRNA (miRNA) sponge]. served as a ceRNA for polo-like kinase (Plk) 2 and cyclin-dependent kinase inhibitor 1 (p21) through binding of miRNA 128 and miRNA 224, inducing Plk2/Nrf2/p21 complexation for Nrf2 activation in the cells under p53-activating conditions (, DNA damage and serum deprivation). expression was suppressed with the initiation of apoptosis, being a rheostat for cell fate determination. levels correlated with the recurrence-free postsurgery survival rate of patients with hepatocellular carcinoma. Collectively, promotes Plk2 and p21 translation by competing for specific miRNAs and activating Nrf2 under surviving conditions from oxidative stress, implying that serves as a fine-tuning rheostat for cell fate decision.-Joo, M. S., Shin, S.-B., Kim, E. J., Koo, J. H., Yim, H., Kim, S. G. Nrf2-lncRNA controls cell fate by modulating p53-dependent Nrf2 activation as an miRNA sponge for Plk2 and p21.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.201802744RDOI Listing
July 2019

LRH1-driven transcription factor circuitry for hepatocyte identity: Super-enhancer cistromic analysis.

EBioMedicine 2019 Feb 9;40:488-503. Epub 2019 Jan 9.

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Republic of Korea. Electronic address:

Background: The injured liver loses normal function, with concomitant decrease of key identity genes. Super-enhancers contribute to mammalian cell identity. Here, we identified core transcription factors (TFs) that are active in hepatocytes, using genome-wide analysis and hierarchical ordering of super-enhancer distribution.

Methods: Expression of core TFs was assessed in a cohort of patients with hepatitis or cirrhosis and animal models. Quantitative PCR, chromatin immunoprecipitation assays, and hydrodynamic gene delivery methods were used to assess gene regulation and hepatocyte viability. RNA-sequencing data were generated to investigate the role of LRH1 in hepatocyte protection from injury.

Results: Network analysis of super-enhancer-associated gene interactions and expression arrays for cohorts of patients with hepatitis and cirrhosis enabled us to identify a super-enhancer-associated network, and LRH1, HNF4α, PPARα, and RXRα as core TFs. In mouse models, expression of core TFs was robustly inhibited by single and multiple challenge(s) with liver toxicant. RNA-seq analysis revealed changes in expression in the super-enhancer-associated genes sensitively biased toward repression by intoxication. LRH1 gene delivery prevented the loss of hepatic super-enhancer-associated signaling circuitry in toxicant-challenged mice, and protected the liver from injury, indicating the role of LRH1 in hepatocyte identity and viability. In hepatocytes, overexpression of each core TF promoted induction of other TFs.

Conclusion: Overall, this study identified LRH1-driven pathway as a circuitry responsible for hepatocyte identity by using cistromic analysis, improving our understanding of liver pathophysiology and identifying novel therapeutic targets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ebiom.2018.12.056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6413675PMC
February 2019

Cell type-dependent function of LATS1/2 in cancer cell growth.

Oncogene 2019 04 10;38(14):2595-2610. Epub 2018 Dec 10.

Department of Pharmacology and Moores Cancer Center, University of California, San Diego, La Jolla, CA, 92093, USA.

The Hippo pathway controls organ size and tissue homeostasis, and its dysregulation often contributes to tumorigenesis. Extensive studies have shown that the Hippo pathway inhibits cell proliferation, and survival in a cell-autonomous manner. We examined the function of the Hippo pathway kinases LATS1/2 (large tumor suppressor 1 and 2) in cancer cells. As expected, loss of LATS1/2 promotes cancer cell growth in most cell lines. Surprisingly, however, LATS1/2 deletion inhibits the growth of murine MC38 colon cancer cells, especially under detachment conditions. This growth inhibitory effect caused by LATS1/2 deletion is due to uncontrolled activation of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), the key downstream transcriptional coactivators inhibited by LATS1/2. We identified Wnt inducible signaling pathway protein 2 (Wisp2) and coiled-coil domain containing 80 (Ccdc80) as direct targets of YAP/TAZ. Their expression is selectively induced by LATS1/2 deletion in MC38 cells. Furthermore, deletion of WISP2 and CCDC80 prevents the growth inhibitory effect of LATS1/2 loss in MC38 cells. Our study demonstrates that the function of LATS1/2 in cell growth is cell context dependent, suggesting that LATS1/2 inhibition can be a therapeutic approach for some cancer types.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41388-018-0610-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450751PMC
April 2019

Gα12 ablation exacerbates liver steatosis and obesity by suppressing USP22/SIRT1-regulated mitochondrial respiration.

J Clin Invest 2018 12 12;128(12):5587-5602. Epub 2018 Nov 12.

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, South Korea.

Nonalcoholic fatty liver disease (NAFLD) arises from mitochondrial dysfunction under sustained imbalance between energy intake and expenditure, but the underlying mechanisms controlling mitochondrial respiration have not been entirely understood. Heterotrimeric G proteins converge with activated GPCRs to modulate cell-signaling pathways to maintain metabolic homeostasis. Here, we investigated the regulatory role of G protein α12 (Gα12) on hepatic lipid metabolism and whole-body energy expenditure in mice. Fasting increased Gα12 levels in mouse liver. Gα12 ablation markedly augmented fasting-induced hepatic fat accumulation. cDNA microarray analysis from Gna12-KO liver revealed that the Gα12-signaling pathway regulated sirtuin 1 (SIRT1) and PPARα, which are responsible for mitochondrial respiration. Defective induction of SIRT1 upon fasting was observed in the liver of Gna12-KO mice, which was reversed by lentivirus-mediated Gα12 overexpression in hepatocytes. Mechanistically, Gα12 stabilized SIRT1 protein through transcriptional induction of ubiquitin-specific peptidase 22 (USP22) via HIF-1α increase. Gα12 levels were markedly diminished in liver biopsies from NAFLD patients. Consistently, Gna12-KO mice fed a high-fat diet displayed greater susceptibility to diet-induced liver steatosis and obesity due to decrease in energy expenditure. Our results demonstrate that Gα12 regulates SIRT1-dependent mitochondrial respiration through HIF-1α-dependent USP22 induction, identifying Gα12 as an upstream molecule that contributes to the regulation of mitochondrial energy expenditure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI97831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264648PMC
December 2018

Interplay between YAP/TAZ and Metabolism.

Cell Metab 2018 08;28(2):196-206

Department of Pharmacology and Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are two homologous transcriptional coactivators that promote cell proliferation, stem cell maintenance, and tissue homeostasis. Under favorable conditions, YAP and TAZ are active to promote cell growth through a transcriptional program mediated by the TEAD family transcription factors. Given the indispensability of cellular energy and metabolites for survival and growth, YAP and TAZ are inhibited when energy level is low. Indeed, glucose, fatty acids, hormones, and other metabolic factors have been recently revealed to regulate YAP and TAZ. Conversely, YAP and TAZ are also involved in metabolism regulation, such as to promote glycolysis, lipogenesis, and glutaminolysis, suggesting YAP and TAZ as emerging nodes in coordinating nutrient availability with cell growth and tissue homeostasis. In this Review, we summarize recent findings and provide a current overview of YAP and TAZ in metabolism by focusing on the role of YAP and TAZ as integrators for metabolic cues and cell growth.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cmet.2018.07.010DOI Listing
August 2018

Cell-Penetrating Function of the Poly(ADP-Ribose) (PAR)-Binding Motif Derived from the PAR-Dependent E3 Ubiquitin Ligase Iduna.

Int J Mol Sci 2018 Mar 8;19(3). Epub 2018 Mar 8.

Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 04763, Korea.

Iduna is a poly(ADP-ribose) (PAR)-dependent E3 ubiquitin ligase that regulates cellular responses such as proteasomal degradation and DNA repair upon interaction with its substrate. We identified a highly cationic region within the PAR-binding motif of Iduna; the region was similar among various species and showed amino acid sequence similarity with that of known cell-penetrating peptides (CPPs). We hypothesized that this Iduna-derived cationic sequence-rich peptide (Iduna) could penetrate the cell membrane and deliver macromolecules into cells. To test this hypothesis, we generated recombinant Iduna-conjugated enhanced green fluorescent protein (Iduna-EGFP) and its tandem-repeat form (d-Iduna-EGFP). Both Iduna-EGFP and d-Iduna-EGFP efficiently penetrated Jurkat cells, with the fluorescence signals increasing dose- and time-dependently. Tandem-repeats of Iduna and other CPPs enhanced intracellular protein delivery efficiency. The delivery mechanism involves lipid-raft-mediated endocytosis following heparan sulfate interaction; d-Iduna-EGFP was localized in the nucleus as well as the cytoplasm, and its residence time was much longer than that of other controls such as TAT and Hph-1. Moreover, following intravenous administration to C57/BL6 mice, d-Iduna-EGFP was efficiently taken up by various tissues, including the liver, spleen, and intestine suggesting that the cell-penetrating function of the human Iduna-derived peptide can be utilized for experimental and therapeutic delivery of macromolecules.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms19030779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5877640PMC
March 2018

Regulation of chitinase-3-like-1 in T cell elicits Th1 and cytotoxic responses to inhibit lung metastasis.

Nat Commun 2018 02 5;9(1):503. Epub 2018 Feb 5.

Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, 04763, Korea.

Chitinase-3-like-1 (Chi3l1) is known to play a significant role in the pathogenesis of Type 2 inflammation and cancer. However, the function of Chi3l1 in T cell and its clinical implications are largely unknown. Here we show that Chi3l1 expression was increased in activated T cells, especially in Th2 cells. In addition, Chi3l1-deficient T cells are hyper-responsive to TcR stimulation and are prone to differentiating into Th1 cells. Chi3l1-deficient Th1 cells show increased expression of anti-tumor immunity genes and decreased Th1 negative regulators. Deletion of Chi3l1 in T cells in mice show reduced melanoma lung metastasis with increased IFNγ and TNFα-producing T cells in the lung. Furthermore, silencing of Chi3l1 expression in the lung using peptide-siRNA complex (dNP2-siChi3l1) efficiently inhibit lung metastasis with enhanced Th1 and CTL responses. Collectively, this study demonstrates Chi3l1 is a regulator of Th1 and CTL which could be a therapeutic target to enhance anti-tumor immunity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-017-02731-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5799380PMC
February 2018

Gα overexpression induced by miR-16 dysregulation contributes to liver fibrosis by promoting autophagy in hepatic stellate cells.

J Hepatol 2018 03 2;68(3):493-504. Epub 2018 Jan 2.

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 08826, Republic of Korea. Electronic address:

Background & Aims: Hepatic stellate cells (HSCs) have a role in liver fibrosis. Guanine nucleotide-binding α-subunit 12 (Gα) converges signals from G-protein-coupled receptors whose ligand levels are elevated in the environment during liver fibrosis; however, information is lacking on the effect of Gα on HSC trans-differentiation. This study investigated the expression of Gα in HSCs and the molecular basis of the effects of its expression on liver fibrosis.

Methods: Gα expression was assessed by immunostaining, and immunoblot analyses of mouse fibrotic liver tissues and primary HSCs. The role of Gα in liver fibrosis was estimated using a toxicant injury mouse model with Gα gene knockout and/or HSC-specific Gα delivery using lentiviral vectors, in addition to primary HSCs and LX-2 cells using microRNA (miR) inhibitors, overexpression vectors, or adenoviruses. miR-16, Gα, and LC3 were also examined in samples from patients with fibrosis.

Results: Gα was overexpressed in activated HSCs and fibrotic liver, and was colocalised with desmin. In a carbon tetrachloride-induced fibrosis mouse model, Gα ablation prevented increases in fibrosis and liver injury. This effect was attenuated by HSC-specific lentiviral delivery of Gα. Moreover, Gα activation promoted autophagy accompanying c-Jun N-terminal kinase-dependent ATG12-5 conjugation. In addition, miR-16 was found to be a direct inhibitor of the de novo synthesis of Gα. Modulations of miR-16 altered autophagy in HSCs. In a fibrosis animal model or patients with severe fibrosis, miR-16 levels were lower than in their corresponding controls. Consistently, cirrhotic patient liver tissues showed Gα and LC3 upregulation in desmin-positive areas.

Conclusions: miR-16 dysregulation in HSCs results in Gα overexpression, which activates HSCs by facilitating autophagy through ATG12-5 formation. This suggests that Gα and its regulatory molecules could serve as targets for the amelioration of liver fibrosis.

Lay Summary: Guanine nucleotide-binding α-subunit 12 (Gα) is upregulated in activated hepatic stellate cells (HSCs) as a consequence of the dysregulation of a specific microRNA that is abundant in HSCs, facilitating the progression of liver fibrosis. This event is mediated by c-Jun N-terminal kinase-dependent ATG12-5 formation and the promotion of autophagy. We suggest that Gα and its associated regulators could serve as new targets in HSCs for the treatment of liver fibrosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jhep.2017.10.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5818314PMC
March 2018

Gα12 regulates osteoclastogenesis by modulating NFATc1 expression.

J Cell Mol Med 2018 02 27;22(2):849-860. Epub 2017 Oct 27.

Department of Cell and Developmental Biology, BK21 Program and Dental Research Institute, Seoul National University, Seoul, Korea.

The G12 family of G protein alpha subunits has been shown to participate in the regulation of various physiological processes. However, the role of Gα12 in bone physiology has not been well described. Here, by micro-CT analysis, we discovered that Gα12-knockout mice have an osteopetrotic phenotype. Histological examination showed lower osteoclast number in femoral tissue of Gα12-knockout mice compared to wild-type mice. Additionally, in vitro osteoclastic differentiation of precursor cells with receptor activator of nuclear factor-κB ligand (RANKL) showed that Gα12 deficiency decreased the number of osteoclast generated and the bone resorption activity. The induction of nuclear factor of activated T-cell c1 (NFATc1), the key transcription factor of osteoclastogenesis, and the activation of RhoA by RANKL was also significantly suppressed by Gα12 deficiency. We further found that the RANKL induction of NFATc1 was not dependent on RhoA signalling, while osteoclast precursor migration and bone resorption required RhoA in the Gα12-mediated regulation of osteoclasts. Therefore, Gα12 plays a role in differentiation through NFATc1 and in cell migration and resorption activity through RhoA during osteoclastogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jcmm.13370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5783869PMC
February 2018

Gα13 ablation reprograms myofibers to oxidative phenotype and enhances whole-body metabolism.

J Clin Invest 2017 Oct 18;127(10):3845-3860. Epub 2017 Sep 18.

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, South Korea.

Skeletal muscle is a key organ in energy homeostasis owing to its high requirement for nutrients. Heterotrimeric G proteins converge signals from cell-surface receptors to potentiate or blunt responses against environmental changes. Here, we show that muscle-specific ablation of Gα13 in mice promotes reprogramming of myofibers to the oxidative type, with resultant increases in mitochondrial biogenesis and cellular respiration. Mechanistically, Gα13 and its downstream effector RhoA suppressed nuclear factor of activated T cells 1 (NFATc1), a chief regulator of myofiber conversion, by increasing Rho-associated kinase 2-mediated (Rock2-mediated) phosphorylation at Ser243. Ser243 phosphorylation of NFATc1 was reduced after exercise, but was higher in obese animals. Consequently, Gα13 ablation in muscles enhanced whole-body energy metabolism and increased insulin sensitivity, thus affording protection from diet-induced obesity and hepatic steatosis. Our results define Gα13 as a switch regulator of myofiber reprogramming, implying that modulations of Gα13 and its downstream effectors in skeletal muscle are a potential therapeutic approach to treating metabolic diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI92067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5617657PMC
October 2017

dNP2-ctCTLA-4 inhibits German cockroach extract-induced allergic airway inflammation and hyper-responsiveness via inhibition of Th2 responses.

Exp Mol Med 2017 08 4;49(8):e362. Epub 2017 Aug 4.

Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, South Korea.

German cockroaches are major household allergens that can trigger allergic airway inflammatory diseases with sensitive T-cell responses. Although the use of immune modulatory biologics, such as antibodies, to mediate allergic responses has recently been examined, only systemic administration is available because of the size limitations on intranasal administration. Here we utilized a cell-permeable peptide, dNP2, to deliver the cytoplasmic domain of cytotoxic T-lymphocyte antigen-4 (ctCTLA-4) through the airway epithelium to modulate Th2 responses in a German cockroach extract (GCE)-induced allergic airway inflammation model. The intranasal delivery efficiency of the dNP2-dTomato protein to the lungs was higher in GCE-induced asthmatic lung parenchymal cells compared to the sham cells. Intranasal administration of the dNP2-ctCTLA-4 protein inhibited airway hyper-responsiveness and reduced airway inflammation and remodeling, including goblet cell metaplasia and collagen deposition around the bronchi. The number of infiltrated cells, including eosinophils, and the levels of IL-4, IL-5, IL-13 and IFN-γ in the lungs were significantly reduced, presumably owing to inhibition of Th2 differentiation. However, intranasal administration of CTLA4-Ig did not inhibit airway inflammation. These results collectively suggest that dNP2-ctCTLA-4 is an efficient intranasally applicable candidate biologic for treating allergic asthma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/emm.2017.107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5579505PMC
August 2017

Oligopeptide Competition Assay for Phosphorylation Site Determination.

J Vis Exp 2017 05 18(123). Epub 2017 May 18.

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University;

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5' adenosine monophosphate-activated protein kinase (AMPK) has been established and was validated using a positional scanning peptide library assay. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3791/55708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608007PMC
May 2017

Protein tyrosine phosphatase conjugated with a novel transdermal delivery peptide, astrotactin 1-derived peptide recombinant protein tyrosine phosphatase (AP-rPTP), alleviates both atopic dermatitis-like and psoriasis-like dermatitis.

J Allergy Clin Immunol 2018 01 26;141(1):137-151. Epub 2017 Apr 26.

Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, Korea; Research Institute for Natural Sciences, Hanyang University, Seoul, Korea; Center for Neuroscience Imaging Research (CNIR), Institute for Basic Science (IBS), Suwon, Korea. Electronic address:

Background: Atopic dermatitis (AD) and psoriasis are the 2 most common chronic inflammatory skin diseases. There is an unmet medical need to overcome limitations for transcutaneous drug development posed by the skin barrier.

Objective: We aimed to identify a novel transdermal delivery peptide and to develop a transcutaneously applicable immunomodulatory protein for treating AD and psoriasis.

Methods: We identified and generated reporter proteins conjugated to astrotactin 1-derived peptide (AP), a novel transdermal delivery peptide of human origin, and analyzed the intracellular delivery efficiency of these proteins in mouse and human skin cells and tissues using multiphoton confocal microscopy. We also generated a recombinant therapeutic protein, AP-recombinant protein tyrosine phosphatase (rPTP), consisting of the phosphatase domain of the T-cell protein tyrosine phosphatase conjugated to AP. The immunomodulatory function of AP-rPTP was confirmed in splenocytes on cytokine stimulation and T-cell receptor stimulation. Finally, we confirmed the in vivo efficacy of AP-rPTP transdermal delivery in patients with oxazolone-induced contact hypersensitivity, ovalbumin-induced AD-like, and imiquimod-induced psoriasis-like skin inflammation models.

Results: AP-conjugated reporter proteins exhibited significant intracellular transduction efficacy in keratinocytes, fibroblasts, and immune cells. In addition, transcutaneous administration of AP-dTomato resulted in significant localization into the dermis and epidermis in both mouse and human skin. AP-rPTP inhibited phosphorylated signal transducer and activator of transcription (STAT) 1, STAT3, and STAT6 in splenocytes and also regulated T-cell activation and proliferation. Transcutaneous administration of AP-rPTP through the paper-patch technique significantly ameliorated skin tissue thickening, inflammation, and cytokine expression in both AD-like and psoriasis-like dermatitis models.

Conclusion: We identified a 9-amino-acid novel transdermal delivery peptide, AP, and demonstrated its feasibility for transcutaneous biologic drug development. Moreover, AP-rPTP is a novel immunomodulatory drug candidate for human dermatitis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jaci.2017.04.007DOI Listing
January 2018

Hepcidin inhibits Smad3 phosphorylation in hepatic stellate cells by impeding ferroportin-mediated regulation of Akt.

Nat Commun 2016 12 22;7:13817. Epub 2016 Dec 22.

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 08826, Korea.

Hepatic stellate cell (HSC) activation on liver injury facilitates fibrosis. Hepatokines affecting HSCs are largely unknown. Here we show that hepcidin inhibits HSC activation and ameliorates liver fibrosis. We observe that hepcidin levels are inversely correlated with exacerbation of fibrosis in patients, and also confirm the relationship in animal models. Adenoviral delivery of hepcidin to mice attenuates liver fibrosis induced by CCl treatment or bile duct ligation. In cell-based assays, either hepcidin from hepatocytes or exogenous hepcidin suppresses HSC activation by inhibiting TGFβ1-mediated Smad3 phosphorylation via Akt. In activated HSCs, ferroportin is upregulated, which can be prevented by hepcidin treatment. Similarly, ferroportin knockdown in HSCs prohibits TGFβ1-inducible Smad3 phosphorylation and increases Akt phosphorylation, whereas ferroportin over-expression has the opposite effect. HSC-specific ferroportin deletion also ameliorates liver fibrosis. In summary, hepcidin suppresses liver fibrosis by impeding TGFβ1-induced Smad3 phosphorylation in HSCs, which depends on Akt activated by a deficiency of ferroportin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms13817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5192182PMC
December 2016

Farnesoid X receptor as a regulator of fuel consumption and mitochondrial function.

Arch Pharm Res 2016 Aug 11;39(8):1062-74. Epub 2016 Aug 11.

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Korea.

Maintenance of energy homeostasis is crucial for survival of organism. There exists a close link between energy metabolism and cell survival, which are coordinately regulated by common signaling pathways. Farnesoid X receptor (FXR) serves as a ligand-mediated transcription factor to regulate diverse genes involved in bile acid, lipid, and glucose metabolism, controlling cellular and systemic energy metabolism. Another important aspect on FXR biology is related to its beneficial effect on cell survival. FXR exerts antioxidative and cytoprotective effect, which is closely associated with the ability of FXR to regulate mitochondrial function. To maintain complex biological processes under homeostasis, FXR activity needs to be dynamically and tightly controlled by different signaling pathways and modifications. In this review, we discuss the role of FXR in the regulation of energy metabolism and cell survival, with the goal of understanding molecular basis for FXR regulation in physiological and pathological conditions. This information may be of assistance in understanding recent advancements of FXR research and strategies for the prevention and treatment of metabolic disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12272-016-0812-yDOI Listing
August 2016

Cell Type Preference of a Novel Human Derived Cell-Permeable Peptide dNP2 and TAT in Murine Splenic Immune Cells.

PLoS One 2016 17;11(5):e0155689. Epub 2016 May 17.

Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, 133-791, Korea.

Cell-permeable peptides (CPPs) have been widely studied as an attractive drug delivery system to deliver therapeutic macromolecules such as DNA, RNA, and protein into cells. However, its clinical application is still limited and controversial due to the lack of a complete understanding of delivery efficiency in target cells. Previously we identified and characterized the novel and superior CPP, named dNP2, and here we comparatively analyzed intracellular delivery efficiency of dNP2 and TAT in various immune cells of mouse spleen to demonstrate their cell type preference. dNP2- or TAT-conjugated fluorescent proteins were most efficiently taken up by phagocytic cells such as dendritic cells and macrophages while little protein uptake was seen by lymphocytes including T cells, B cells, and NK cells. Interestingly CD8+ lymphoid dendritic cells and CD62LloCD44hi memory like T cell subsets showed significantly better uptake efficiency in vitro and in vivo relative to other dendritic cells or T cells, respectively. In addition, activated macrophages, T cells, and B cells took up the proteins more efficiently relative to when in the resting state. Importantly, only dNP2, not TAT, shows significant intracellular protein delivery efficiency in vivo. Collectively, this study provides important information regarding heterogeneous intracellular delivery efficiency of CPPs such as dNP2 and TAT with cell type preference in the spleen needed for its application in phagocytic cells or activated immune cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0155689PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4871486PMC
July 2017

AMPK Facilitates Nuclear Accumulation of Nrf2 by Phosphorylating at Serine 550.

Mol Cell Biol 2016 07 29;36(14):1931-42. Epub 2016 Jun 29.

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, South Korea

Nrf2 (nuclear factor erythroid 2-related factor 2) is an antioxidant transcription factor. AMP-activated protein kinase (AMPK) functions as a central regulator of cell survival in response to stressful stimuli. Nrf2 should be coordinated with the cell survival pathway controlled by AMPK, but so far the mechanistic connections remain undefined. This study investigated the role of AMPK in Nrf2 trafficking and its activity regulation. A subnetwork integrating neighbor molecules suggested direct interaction between AMPK and Nrf2. In cells, AMPK activation caused nuclear accumulation of Nrf2. In the in vitro kinase and peptide competition assays, AMPK phosphorylated Nrf2 at the Ser558 residue (Ser550 in mouse) located in the canonical nuclear export signal. Nrf2 with an S550A mutation failed to be accumulated in the nucleus after AMPK activation. Leptomycin B, a nuclear export inhibitor, did not enhance nuclear accumulation of wild-type Nrf2 (WT-Nrf2) activated by AMPK or a phospho-Ser550-mimetic Nrf2 mutant, corroborating the finding that AMPK facilitated nuclear accumulation of Nrf2, probably by inhibiting nuclear export. Activated glycogen synthase kinase 3β (GSK3β) diminished the basal nuclear level of Myc-S550A-Nrf2. Taking the data collectively, AMPK phosphorylates Nrf2 at the Ser550 residue, which, in conjunction with AMPK-mediated GSK3β inhibition, promotes nuclear accumulation of Nrf2 for antioxidant response element (ARE)-driven gene transactivation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.00118-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4936058PMC
July 2016

Use of Cell-Penetrating Peptides in Dendritic Cell-Based Vaccination.

Immune Netw 2016 Feb 25;16(1):33-43. Epub 2016 Feb 25.

Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 04763, Korea.; Research Institute for Natural Sciences, Hanyang University, Seoul 04763, Korea.

Cell-penetrating peptides (CPPs) are short amino acids that have been widely used to deliver macromolecules such as proteins, peptides, DNA, or RNA, to control cellular behavior for therapeutic purposes. CPPs have been used to treat immunological diseases through the delivery of immune modulatory molecules in vivo. Their intracellular delivery efficiency is highly synergistic with the cellular characteristics of the dendritic cells (DCs), which actively uptake foreign antigens. DC-based vaccines are primarily generated by pulsing DCs ex vivo with various immunomodulatory antigens. CPP conjugation to antigens would increase DC uptake as well as antigen processing and presentation on both MHC class II and MHC class I molecules, leading to antigen specific CD4(+) and CD8(+) T cell responses. CPP-antigen based DC vaccination is considered a promising tool for cancer immunotherapy due to the enhanced CTL response. In this review, we discuss the various applications of CPPs in immune modulation and DC vaccination, and highlight the advantages and limitations of the current CPP-based DC vaccination.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4110/in.2016.16.1.33DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4770098PMC
February 2016

Endoplasmic Reticulum Stress in Hepatic Stellate Cells Promotes Liver Fibrosis via PERK-Mediated Degradation of HNRNPA1 and Up-regulation of SMAD2.

Gastroenterology 2016 Jan 3;150(1):181-193.e8. Epub 2015 Oct 3.

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Korea. Electronic address:

Background & Aims: Endoplasmic reticulum (ER) stress has been implicated in a variety of diseases. Hepatic stellate cells (HSCs) contribute to the development of liver fibrosis. Information on the link between ER stress and HSC activation is scarce. We investigated the effects of ER stress in HSCs on the progression of liver fibrosis and the regulation of this process in cells and mice.

Methods: Proteins and messenger RNAs were measured in 2 sets of liver samples (n = 25 and n = 44) collected from patients with chronic hepatitis C virus infection and/or fibrosis. ER stress was induced in cells and mice using chemical agents. Lentiviral vectors were constructed to express glucose-regulated protein 78 (GRP78; also known as HSPA5) or heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) from the α-smooth muscle actin promoter and injected into C57BL/6 mice for HSC-specific gene expression. Liver tissues and HSCs were collected from mice or rats and analyzed using immunoblottings and quantitative reverse-transcription polymerase chain reaction. LX-2 cells were transfected with small interfering RNAs, microRNA mimics, or overexpression vectors.

Results: Hepatic ER stress was much higher in liver tissues from patients with severe vs mild fibrosis. ER stress induced fibrogenic genes in HSCs. Targeted lentiviral delivery of glucose-regulated protein 78 to HSCs in mice reduced fiber accumulation in liver. Levels of SMAD2, but not SMAD3, were increased in fibrotic liver tissues from patients or mice exposed to ER stress; small interfering RNA-mediated knockdown of SMAD2 reduced ER stress-mediated activation of HSCs. In rat HSCs, ER stress increased levels of SMAD2 messenger RNA by decreasing levels of microRNA 18a (MIR18A), an inhibitor of SMAD2 expression, rather than transactivating the SMAD2 gene. ER stress-activated PKR-like endoplasmic reticulum kinase, also known as EIF2AK3 (PERK) phosphorylated HNRNPA1, a protein required for the maturational processing of primary MIR18A, at Thr51, accelerating its degradation. Overexpression of HNRNPA1 (or its T51A mutant) in HSCs of mice inhibited liver fibrosis. Severe fibrotic liver tissues from patients had increased levels of phosphorylated PERK and reduced levels of HNRNPA1 in HSCs, compared with mild fibrotic liver tissues.

Conclusions: ER stress in HSCs promotes liver fibrosis by inducing overexpression of SMAD2, via dysregulation of MIR18A; this dysregulation is mediated by PERK phosphorylation and destabilization of HNRNPA1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1053/j.gastro.2015.09.039DOI Listing
January 2016

dNP2 is a blood-brain barrier-permeable peptide enabling ctCTLA-4 protein delivery to ameliorate experimental autoimmune encephalomyelitis.

Nat Commun 2015 Sep 15;6:8244. Epub 2015 Sep 15.

Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791, Republic of Korea.

Central nervous system (CNS)-infiltrating effector T cells play critical roles in the development and progression of multiple sclerosis (MS). However, current drugs for MS are very limited due to the difficulty of delivering drugs into the CNS. Here we identify a cell-permeable peptide, dNP2, which efficiently delivers proteins into mouse and human T cells, as well as various tissues. Moreover, it enters the brain tissue and resident cells through blood vessels by penetrating the tightly organized blood-brain barrier. The dNP2-conjugated cytoplasmic domain of cytotoxic T-lymphocyte antigen 4 (dNP2-ctCTLA-4) negatively regulates activated T cells and shows inhibitory effects on experimental autoimmune encephalomyelitis in both preventive and therapeutic mouse models, resulting in the reduction of demyelination and CNS-infiltrating T helper 1 and T helper 17 cells. Thus, this study demonstrates that dNP2 is a blood-brain barrier-permeable peptide and dNP2-ctCTLA-4 could be an effective agent for treating CNS inflammatory diseases such as MS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms9244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4579786PMC
September 2015
-->