Development of a Label-Free LC-MS/MS-Based Glucosylceramide Synthase Assay and Its Application to Inhibitors Screening for Ceramide-Related Diseases

Zhicheng Fu,윤소윤,원종훈,백문정,장지민,하해찬,이해경,신인철,김주은,김희수,김대경 한국응용약물학회 2019 Biomolecules & Therapeutics(구 응용약물학회지) Vol.27 No.2

Ceramide metabolism is known to be an essential etiology for various diseases, such as atopic dermatitis and Gaucher disease. Glucosylceramide synthase (GCS) is a key enzyme for the synthesis of glucosylceramide (GlcCer), which is a main ceramide metabolism pathway in mammalian cells. In this article, we developed a liquid chromatography-tandem mass spectrometry (LCMS/ MS) method to determine GCS activity using synthetic non-natural sphingolipid C8-ceramide as a substrate. The reaction products, C8-GlcCer for GCS, could be separated on a C18 column by reverse-phase high-performance liquid chromatography (HPLC). Quantification was conducted using the multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 588.6 → 264.4 for C8-GlcCer at positive ionization mode. The calibration curve was established over the range of 0.625-160 ng/mL, and the correlation coefficient was larger than 0.999. This method was successfully applied to detect GCS in the human hepatocellular carcinoma cell line (HepG2 cells) and mouse peripheral blood mononuclear cells. We also evaluated the inhibition degree of a known GCS inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on GCS enzymatic activity and proved that this method could be successfully applied to GCS inhibitor screening of preventive and therapeutic drugs for ceramide metabolism diseases, such as atopic dermatitis and Gaucher disease.

Theories of Religious Relationship and Christian-Buddhist Dialogue : A Christian Perspective

Wang Zhicheng,Fu Yu 장로회신학대학교 2014 ASIA‒PACIFIC JOURNAL OF THEOLOGICAL STUDIES Vol.2 No.-

This paper aims to examine the relationship between Buddhism and Christianity, especially the issue of how to see “the Other” through relationship theories of modern religions, e.g. Exclusivism, Inclusivism, Particularism, Pluralism and Comparativism. The exclusivists believe that conversion to Christianity is the only way for other religious adherents to salvation or liberation, thereby they deny the existence of identity among religious others, rendering the Christian-Buddhist Dialogue (CBD) impossible; the variant of Exclusivism-Particularism seeks to avoid the above impasse by making some room for dialogue through the distinction between language systems, the Otherness was translated into incommensurability arising from different languages; the Inclusivists subsume the Other as “Anonymous Christians” so that CBD turns into certain form of internal talk, the religious Others lose their own identities in this way; the pluralists respect the Other and their identities, then CBD enhances mutual understanding and enrichment for dialogue partners; the comparativists accept the Other and tries to go across the boundaries of religions, to be immered in the Other tradition and learn from the Other in the Other’s terminology and perspective. In this way, the understanding of one’s own tradition and faith are reinforced from the new insights.

Protective effect of sesquiterpene lactone parthenolide on LPS-induced acute lung injury

장유진,백문정,Zhicheng Fu,이주현,원종훈,하해찬,이혜경,장지민,최종민,김대경 대한약학회 2016 Archives of Pharmacal Research Vol.39 No.12

Acute lung injury (ALI) is a respiratory failuredisease and the major source of mortality in the critically illpatients. The main pathological changes involved in ALIinclude the excessive recruitment and activation of neutrophilsby increased pro-inflammatory mediators. However,any specific therapy for ALI has not been developed. The objective of this study was to investigate protectiveeffects of parthenolide, a sesquiterpene lactone produced infeverfew, on LPS-induced lung injury. In the present study,parthenolide treatment reduced infiltration of inflammatorycells, airway permeability and production of pro-inflammatorycytokines in LPS-induced ALI mouse model. Further,LPS-stimulated phosphorylation of NF-jB, the keyregulatory transcription factor in ALI, was inhibited byparthenolide treatment in lung epithelial BEAS-2B cellsand alveolar macrophage MH-S cells. These results suggestthat parthenolide may provide a beneficial therapeuticstrategy for ALI.

Paraquat Induces Apoptosis through a Mitochondria-Dependent Pathway in RAW264.7 Cells

장여진,원종훈,백문정,Zhicheng Fu,장지민,하해찬,홍승범,장민선,김대경 한국응용약물학회 2015 Biomolecules & Therapeutics(구 응용약물학회지) Vol.23 No.5

Paraquat dichloride (N,N-dimethyl-4-4′-bipiridinium, PQ) is an extremely toxic chemical that is widely used in herbicides. PQ generates reactive oxygen species (ROS) and causes multiple organ failure. In particular, PQ has been reported to be an immunotoxic agrochemical compound. PQ was shown to decrease the number of macrophages in rats and suppress monocyte phagocytic activity in mice. However, the effect of PQ on macrophage cell viability remains unclear. In this study, we evaluated the cytotoxic effect of PQ on the mouse macrophage cell line, RAW264.7 and its possible mechanism of action. RAW264.7 cells were treated with PQ (0, 75, and 150 μM), and cellular apoptosis, mitochondrial membrane potential (MMP), and intracellular ROS levels were determined. Morphological changes to the cell nucleus and cellular apoptosis were also evaluated by DAPI and Annexin V staining, respectively. In this study, PQ induced apoptotic cell death by dose-dependently decreasing MMP. Additionally, PQ increased the cleaved form of caspase-3, an apoptotic marker. In conclusion, PQ induces apoptosis in RAW264.7 cells through a ROS-mediated mitochondrial pathway. Thus, our study improves our knowledge of PQ-induced toxicity, and may give us a greater understanding of how PQ affects the immune system.

Novel Effect of Hyaluronan and Proteoglycan Link Protein 1 (HAPLN1) on Hair Follicle Cells Proliferation and Hair Growth

Ha Hae Chan,Zhou Dan,Fu Zhicheng,Back Moon Jung,Jang Ji Min,Shin In Chul,Kim Dae Kyong 한국응용약물학회 2023 Biomolecules & Therapeutics(구 응용약물학회지) Vol.31 No.5

Hair loss is a common condition that can have a negative impact on an individual’s quality of life. The severe side effects and the low efficacy of current hair loss medications create unmet needs in the field of hair loss treatment. Hyaluronan and Proteoglycan Link Protein 1 (HAPLN1), one of the components of the extracellular matrix, has been shown to play a role in maintaining its integrity. HAPLN1 was examined for its ability to impact hair growth with less side effects than existing hair loss treatments. HAPLN1 was predominantly expressed in the anagen phase in three stages of the hair growth cycle in mice and promotes the proliferation of human hair matrix cells. Also, recombinant human HAPLN1 (rhHAPLN1) was shown to selectively increase the levels of transforming growth factor-β receptor II in human hair matrix cells. Furthermore, we observed concomitant activation of the ERK1/2 signaling pathway following treatment with rhHAPLN1. Our results indicate that rhHAPLN1 elicits its cell proliferation effect via the TGF-β2-induced ERK1/2 pathway. The prompt entering of the hair follicles into the anagen phase was observed in the rhHAPLN1- treated group, compared to the vehicle-treated group. Insights into the mechanism underlying such hair growth effects of HAPLN1 will provide a novel potential strategy for treating hair loss with much lower side effects than the current treatments.

Recombinant Human HAPLN1 Mitigates Pulmonary Emphysema by Increasing TGF-β Receptor I and Sirtuins Levels in Human Alveolar Epithelial Cells

김대경,Yongwei Piao,So Yoon Yun,Zhicheng Fu,Ji Min Jang,Moon Jung Back,Ha Hyung Kim 한국분자세포생물학회 2023 Molecules and cells Vol.46 No.9

Chronic obstructive pulmonary disease (COPD) will be the third leading cause of death worldwide by 2030. One of its components, emphysema, has been defined as a lung disease that irreversibly damages the lungs’ alveoli. Treatment is currently unavailable for emphysema symptoms and complete cure of the disease. Hyaluronan (HA) and proteoglycan link protein 1 (HAPLN1), an HA-binding protein linking HA in the extracellular matrix to stabilize the proteoglycan structure, forms a bulky hydrogel-like aggregate. Studies on the biological role of the full-length HAPLN1, a simple structure-stabilizing protein, are limited. Here, we demonstrated for the first time that treating human alveolar epithelial type 2 cells with recombinant human HAPLN1 (rhHAPLN1) increased TGF-β receptor 1 (TGF-β RI) protein levels, but not TGF-β RII, in a CD44-dependent manner with concurrent enhancement of the phosphorylated Smad3 (p-Smad3), but not p-Smad2, upon TGF-β1 stimulation. Furthermore, rhHAPLN1 significantly increased sirtuins levels (i.e., SIRT1/2/6) without TGF-β1 and inhibited acetylated p300 levels that were increased by TGF-β1. rhHAPLN1 is crucial in regulating cellular senescence, including p53, p21, and p16, and inflammation markers such as p-NF-κB and Nrf2. Both senile emphysema mouse model induced via intraperitoneal rhHAPLN1 injections and porcine pancreatic elastase (PPE)-induced COPD mouse model generated via rhHAPLN1-containing aerosols inhalations showed a significantly potent efficacy in reducing alveolar spaces enlargement. Preclinical trials are underway to investigate the effects of inhaled rhHAPLN1-containing aerosols on several COPD animal models.

Paraquat Induces Apoptosis through a Mitochondria-Dependent Pathway in RAW264.7 Cells

Jang, Yeo Jin,Won, Jong Hoon,Back, Moon Jung,Fu, Zhicheng,Jang, Ji Min,Ha, Hae Chan,Hong, SeungBeom,Chang, Minsun,Kim, Dae Kyong The Korean Society of Applied Pharmacology 2015 Biomolecules & Therapeutics(구 응용약물학회지) Vol.23 No.5

Paraquat dichloride (N,N-dimethyl-4-4'-bipiridinium, PQ) is an extremely toxic chemical that is widely used in herbicides. PQ generates reactive oxygen species (ROS) and causes multiple organ failure. In particular, PQ has been reported to be an immunotoxic agrochemical compound. PQ was shown to decrease the number of macrophages in rats and suppress monocyte phagocytic activity in mice. However, the effect of PQ on macrophage cell viability remains unclear. In this study, we evaluated the cytotoxic effect of PQ on the mouse macrophage cell line, RAW264.7 and its possible mechanism of action. RAW264.7 cells were treated with PQ (0, 75, and $150{\mu}M$), and cellular apoptosis, mitochondrial membrane potential (MMP), and intracellular ROS levels were determined. Morphological changes to the cell nucleus and cellular apoptosis were also evaluated by DAPI and Annexin V staining, respectively. In this study, PQ induced apoptotic cell death by dose-dependently decreasing MMP. Additionally, PQ increased the cleaved form of caspase-3, an apoptotic marker. In conclusion, PQ induces apoptosis in RAW264.7 cells through a ROS-mediated mitochondrial pathway. Thus, our study improves our knowledge of PQ-induced toxicity, and may give us a greater understanding of how PQ affects the immune system.

A Novel Role of Hyaluronic Acid and Proteoglycan Link Protein 1 (HAPLN1) in Delaying Vascular Endothelial Cell Senescence

Zhou Dan,Jang Ji Min,Yang Goowon,Ha Hae Chan,Fu Zhicheng,Kim Dae Kyong 한국응용약물학회 2023 Biomolecules & Therapeutics(구 응용약물학회지) Vol.31 No.6

Cardiovascular diseases (CVDs) are the most common cardiovascular system disorders. Cellular senescence is a key mechanism associated with dysfunction of aged vascular endothelium. Hyaluronic acid and proteoglycan link protein 1 (HAPLN1) has been known to non-covalently link hyaluronic acid (HA) and proteoglycans (PGs), and forms and stabilizes HAPLN1-containing aggregates as a major component of extracellular matrix. Our previous study showed that serum levels of HAPLN1 decrease with aging. Here, we found that the HAPLN1 gene expression was reduced in senescent human umbilical vein endothelial cells (HUVECs). Moreover, a recombinant human HAPLN1 (rhHAPLN1) decreased the activity of senescence-associated β-gal and inhibited the production of senescence-associated secretory phenotypes, including IL-1β, CCL2, and IL-6. rhHAPLN1 also downregulated IL-17A levels, which is known to play a key role in vascular endothelial senescence. In addition, rhHAPLN1 protected senescent HUVECs from oxidative stress by reducing cellular reactive oxygen species levels, thus promoting the function and survival of HUVECs and leading to cellular proliferation, migration, and angiogenesis. We also found that rhHAPLN1 not only increases the sirtuin 1 (SIRT1) levels, but also reduces the cellular senescence markers levels, such as p53, p21, and p16. Taken together, our data indicate that rhHAPLN1 delays or inhibits the endothelial senescence induced by various aging factors, such as replicative, IL-17A, and oxidative stress-induced senescence, thus suggesting that rhHAPLN1 may be a promising therapeutic for CVD and atherosclerosis.

Hyaluronic acid and proteoglycan link protein 1 suppresses platelet‑derived growth factor-BB-induced proliferation, migration, and phenotypic switching of vascular smooth muscle cells

주단,Hae Chan Ha,양구원,Ji Min Jang,박보경,Zhicheng Fu,In Chul Shin,김대경 생화학분자생물학회 2023 BMB Reports Vol.56 No.8

The development of atherosclerotic cardiovascular disease isassociated with the phenotypic switching of vascular smoothmuscle cells (SMCs) from a contractile to a synthetic state,leading to cell migration and proliferation. Platelet‑derivedgrowth factor‑BB (PDGF‑BB) modulates this de-differentiationby initiating a number of biological processes. In this study,we show that gene expression of hyaluronic acid (HA) andproteoglycan link protein 1 (HAPLN1) was upregulated duringdifferentiation of human aortic SMCs (HASMCs) into a contractilestate, but downregulated upon during PDGF-BB-induced dedifferentiation. This is the first study showing that the treatmentof HASMCs with full-length recombinant human HAPLN1(rhHAPLN1) significantly reversed PDGF-BB-induced decreasein the protein levels of contractile markers (SM22α, α-SMA,calponin, and SM-MHC), and inhibited the proliferation andmigration of HASMCs induced by PDGF-BB. Furthermore, ourresults show that rhHAPLN1 significantly inhibited thephosphorylation of FAK, AKT, STAT3, p38 MAPK and Rafmediated by the binding of PDGF-BB to PDGFRβ. Together,these results indicated that rhHAPLN1 can suppress thePDGF-BB-stimulated phenotypic switching and subsequentde-differentiation of HASMCs, highlighting its potential as anovel therapeutic target for atherosclerosis and other vasculardiseases.

A Novel Therapeutic Effect of a New Variant of CTLA4-Ig with Four Antennas That Are Terminally Capped with Sialic Acid in the CTLA4 Region

Piao Yongwei,Yun So Yoon,Kim Hee Soo,Park Bo Kyung,Ha Hae Chan,Fu Zhicheng,Jang Ji Min,Back Moon Jung,Shin In Chul,Won Jong Hoon,Kim Dae Kyong 한국응용약물학회 2022 Biomolecules & Therapeutics(구 응용약물학회지) Vol.30 No.6

Rheumatoid arthritis (RA) is a multifactorial immune-mediated disease, the pathogenesis of which involves different cell types. T-cell activation plays an important role in RA. Therefore, inhibiting T-cell activation is one of the current therapeutic strategies. Cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4-Ig), also known as abatacept, reduces cytokine secretion by inhibiting T-cell activation. To achieve a homeostatic therapeutic effect, CTLA4-Ig has to be administered repeatedly over several weeks, which limits its applicability in RA treatment. To overcome this limitation, we increased the number of sialic acid-capped antennas by genetically engineering the CTLA4 region to increase the therapeutic effect of CTLA4-Ig. N-acetylglucosaminyltransferase (GnT) and α2,6-sialyltransferase (α2,6-ST) were co-overexpressed in Chinese hamster ovary (CHO) cells to generate a highly sialylated CTLA4-Ig fusion protein, named ST6. The therapeutic and immunogenic effects of ST6 and CTLA4-Ig were compared. ST6 dose-dependently decreased paw edema in a mouse model of collagen-induced arthritis and reduced cytokine levels in a co-culture cell assay in a similar manner to CTLA4-Ig. ST6- and CTLA4-Ig-induced T cell-derived cytokines were examined in CD4 T cells isolated from peripheral blood mononuclear cells after cell killing through irradiation followed by flow- and magnetic-beadassisted separation. Interestingly, compared to CTLA4-Ig, ST6 was substantially less immunogenic and more stable and durable. Our data suggest that ST6 can serve as a novel, less immunogenic therapeutic strategy for patients with RA.