cDNA Library를 이용한 생쥐 두개골 유래 조골세포의 유전자 발현 분석

정연관,정재환,최제용 대한골대사학회 2008 대한골대사학회지 Vol.15 No.1

Background: Large-scale sequencing of cDNA clones from cDNA library is a powerful approach for the discovery of novel genes, identification of novel gene family members and definition of gene expression profiles. Listing up of bone related genes is necessary for better understanding of molecular bone physiology and homeostasis. Methods: Primary cultured mouse calvarial osteoblast (MCO) was isolated from mouse embryo at day 17.5. Total RNA was purified from MCO and constructed cDNA library using Stratagen cDNA library contstruction kit. Random selected clones of cDNA library were purified and analyzed the sequence by sequencing analysis. Sequences were identified using BlastN in NCBI mouse EST database. Results: The high quality of MCO cDNA library was constructed showing low background (7.2%) such as no insert clone, mitochondrial DNA, E. coli gene, etc. Total 2361 clones were identified by random sequencing. Except low sequencing quality (207), 2154 sequences were further analyzed. In analyzed 1999 useable clones, 1137 clones were known gene, 231 clones were similar to known genes, 69 clones were novel genes, and 562 clones were expression sequence tag (EST). Majority of identified 1078 clones were detected only once, and the others were over two times. The most abundant genes were type I collagen, α2(I) and α1(I) that were detected 36 and 32 times each. Also osteopontin, actin, Thbs1, Fth and Lgals1 were detected highly. Conclusion: In NCBI EST library database, there is only one library related to mouse osteoblasts. Our results will provide a framework data for understanding the gene expression in osteoblasts.

Osteoclasts in the Inflammatory Arthritis: Implications for Pathologic Osteolysis

정연관,강영모,한승우 대한면역학회 2019 Immune Network Vol.19 No.1

The enhanced differentiation and activation of osteoclasts (OCs) in the inflammatory arthritis such as rheumatoid arthritis (RA) and gout causes not only local bone erosion, but also systemic osteoporosis, leading to functional disabilities and morbidity. The induction and amplification of NFATc1, a master regulator of OC differentiation, is mainly regulated by receptor activator of NF-κB (RANK) ligand-RANK and calcium signaling which are amplified in the inflammatory milieu, as well as by inflammatory cytokines such as TNFα, IL-1β and IL-6. Moreover, the predominance of CD4+ T cell subsets, which varies depending on the condition of inflammatory diseases, can determine the fate of OC differentiation. Anti-citrullinated peptide antibodies which are critical in the pathogenesis of RA can bind to the citrullinated vimentin on the surface of OC precursors, and in turn promote OC differentiation and function via IL-8. In addition to adaptive immunity, the activation of innate immune system including the nucleotide oligomerization domain leucine rich repeat with a pyrin domain 3 inflammasome and TLRs can regulate OC maturation. The emerging perspectives about the diverse and close interactions between the immune cells and OCs in inflammatory milieu can have a significant impact on the future direction of drug development.

DICAM에 의한 LPS-매개 대식세포 활성화 억제

정연관 ( Youn Kwan Jung ),박혜리 ( Hye Ri Park ),이은주 ( Eun Ju Lee ),정동형 ( Dong Hyoung Jeong ),김건우 ( Gun Woo Kim ),최제용 ( Je Yong Choi ),한승우 ( Seung Woo Han ) 대한류마티스학회 2012 대한류마티스학회지 Vol.19 No.4

DICAM, a dual Ig domain containing adhesion molecule, is involved in cell-cell adhesion through direct interaction with αvβ3 integrin. In our previous study showing the inhibitory role of DICAM in osteoclast differentiation, we found that DICAM also has a suppressive role in macrophage, the precursor cell of osteoclast, The role of DICAM in macrophage activation at the inflammatory milieu, however, remains obscure, Methods, Expression pattern of DICAM by inflammatory cytokines and lipopolysaccharide (LPS) was studied with RAW264.7, a murine macrophage cell line, To study the role of DICAM on macrophage activation, we stably transduced DICAM, or empty vector, into RAW264.7, and then compared the LPS-mediated activation such as spreading and TNF-a production, Results, DICAM was abundantly expressed in the synovial tissue of collagen-induced arthritis. When we assessed the expression of DICAM in RAW264.7 cells by mediators of inflammation, inflammatory cytokines, such as TNF-α, IL-1β, and IFN-γ, and M-CSF increased the expression of DICAM; however, LPS decreased. Functionally, DICAM that stably transduced-RAW264.7 cells showed attenuation of LPS-mediated macrophage activation including spreading and TNF-α production. DICAM decreased the phosphorylation of JNK MAP kinase by M-CSF and LPS stimulation, which was corroborated by a decrease in the expression of ITAM-associated receptors including Trem2, Pira1, and Oscar. Finally, a recombinant ectodomain of DICAM suppressed LPS-induced activation of RAW264.7 cells, Conclusion. These results indicate that DICAM acts as a negative regulator of LPS-mediated macrophage activation.

Two Types of Mouse Models for Sarcopenia Research: Senescence Acceleration and Genetic Modification Models

백경완,정연관,박진성,김지석,하영술,김소정,유준일 대한골대사학회 2021 대한골대사학회지 Vol.28 No.3

Sarcopenia leads to loss of skeletal muscle mass, quality, and strength due to aging; it was recently given a disease code (International Classification of Diseases, Tenth Revision, Clinical Modification, M62.84). As a result, in recent years, sarcopenia-related research has increased. In addition, various studies seeking to prevent and treat sarcopenia by identifying the various mechanisms related to the reduction of skeletal muscle properties have been conducted. Previous studies have identified muscle synthesis and breakdown; investigating them has generated evidence for preventing and treating sarcopenia. Mouse models are still the most useful ones for determining mechanisms underlying sarcopenia through correlations and interventions involving specific genes and their phenotypes. Mouse models used to study sarcopenia often induce muscle atrophy by hindlimb unloading, denervation, or immobilization. Though it is less frequently used, the senescence-accelerated mouse can also be useful for sarcopenia research. Herein, we discuss cases where senescence-accelerated and genetically engineered mouse models were used in sarcopenia research and different perspectives to use them.

P-126 Sporadic primary pulmonary hypertension results from a novel mutation in the bone morphogenic protein receptor-II gene

최선하,박재석,정연관,한승우,정병천 대한결핵 및 호흡기학회 2017 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.124 No.0

Most common genetic cause of primary pulmonary hypertension (PAH) has been ascribed to heterozygous germline mutations in the bone morphogenic protein receptor-II (BMPR2) gene which account for approximately 10 to 40 % of apparently sporadic form and in 58 to 74 % of patients with familial PAH. Here we report a novel frameshift mutation of BMPR2 identified in a sporadic PAH patient. A 22-year-old female with one month history of progressive dyspnea associated with central localised chest pain. An echocardiogram showed severe right ventricle dilatation and hypertrophy with moderate RV systolic dysfunction in addition to a huge right atrium and severe tricuspid regurgitation. Right ventricle systolic pressure was 86 mmHg with no evidence of left to right shunt with negative contrast injection. Coronary angiography showed no significant stenosis of the coronary arteries. Direct sequencing of the BMPR2 gene revealed heterozygous frameshift mutation in exon 2 that an additional T allele is inserted in the cDNA position of 1263 (NM_001204) resulting in early stop codon at the amino acid position 48 (c.1263InsT: p.Y40fsX48). BMPR2 expression in the peripheral blood mononuclear cells (PBMCs) of patient was nearly half of control in protein level, while BMPR2 RNA was increased about 10-fold in the patient’s PBMCs compared to controls. When treated with PMA, TGFβ, and BMP2, the PBMCs from patient showed an aberrantly enhanced phosphorylation of Smad2/3, but that of Smad1/5 was decreased, suggesting imbalance of transforming growth factor-β (TGFβ) and BMP signaling.

Rodent Model of Muscular Atrophy for Sarcopenia Study

Kyung-Wan Baek,정연관,Ji-Seok Kim,박진성,하영술,So-Jeong Kim,유준일 대한골대사학회 2020 대한골대사학회지 Vol.27 No.2

The hallmark symptom of sarcopenia is the loss of muscle mass and strength without the loss of overall body weight. Sarcopenia patients are likely to have worse clinical outcomes and higher mortality than do healthy individuals. The sarcopenia population shows an annual increase of ~0.8% in the population after age 50, and the prevalence rate is rapidly increasing with the recent worldwide aging trend. Based on International Classification of Diseases, Tenth Revision, a global classification of disease published by the World Health Organization, issued the disease code (M62.84) given to sarcopenia in 2016. Therefore, it is expected that the study of sarcopenia will be further activated based on the classification of disease codes in the aging society. Several epidemiological studies and meta-analyses have looked at the correlation between the prevalence of sarcopenia and several environmental factors. In addition, studies using cell lines and rodents have been done to understand the biological mechanism of sarcopenia. Laboratory rodent models are widely applicable in sarcopenia studies because of the advantages of time savings, cost saving, and various analytical applications that could not be used for human subjects. The rodent models that can be applied to the sarcopenia research are diverse, but a simple and fast method that can cause atrophy or aging is preferred. Therefore, we will introduce various methods of inducing muscular atrophy in rodent models to be applied to the study of sarcopenia.

THP-1 세포주의 대식세포 분화과정에서 DICAM에 의한 제 1형 인터페론 시스템의 억제

김보연 ( Bo Yeon Kim ),박인 ( In Park ),정연관 ( Youn Kwan Jung ),한민수 ( Min Su Han ),김건우 ( Gun Woo Kim ),한승우 ( Seung Woo Han ) 대한류마티스학회 2014 대한류마티스학회지 Vol.21 No.3

Objective. We have previously shown that DICAM inhibits LPS-mediated macrophage differentiation. However, less is known about the exact action mechanisms of DICAM on the macrophage function and differentiation. Methods. To induce differentiation into a resting M0 macrophage, THP-1 cells were cultured with 100 nM PMA for 24 h, and then rested for 3 days. THP-1 cells were infected with 50 moi of control LacZ- or DICAM-containing adenovirus. The RNA expression profile associated with DICAM during THP-1 differentiation was analyzed with a microarray chip and in silico analysis with Ingenuity Pathway Analysis (IPA) program. Results. A disease and function analysis of the microarray data in DICAM-overexpressed THP-1 cells revealed a suppression in the expression of multiple genes involved in the response of myeloid cells and phagocytes, and an increase of genes associated with apoptosis of fibroblast cell-line, and viral infection and replication. The canonical pathway analysis also showed the most prominent changes of signaling pathways that involve inflammation responses. An upstream regulator analysis identifyingmolecules upstream of the genes that potentially explain the observed expression changes revealed that IRF7 and the genes in type 1 interferon system, such as IFNA2 and IFNAR,was significantly attenuated by DICAM. A mechanistic network analysis confirmed a direct causal association between IRF7 and type 1 interferon system. A real-time RT-PCR analysis validating the microarray data verified the significant suppression of IRFs, IFNA2, and IFNB1. Conclusion. These results suggest that DICAM can be a critical regulator of type 1 interferon system, which is an essential mediator in the process of intracellular infection and systemic lupus erythematosus.