자궁 내 배아 착상 환경에 대한 이해

송행석,Song, Haengseok 대한생식의학회 2006 Clinical and Experimental Reproductive Medicine Vol.33 No.3

Cadmium-induced ER stress and inflammation are mediated through C/EBP–DDIT3 signaling in human bronchial epithelial cells

김지영,송행석,허혜련,김정운,김혜련,홍윤기,양세란,한선숙,이승준,김우진,홍석호 생화학분자생물학회 2017 Experimental and molecular medicine Vol.49 No.-

Cadmium (Cd), a major component of cigarette smoke, disrupts the normal functions of airway cells and can lead to the development of various pulmonary diseases such as chronic obstructive pulmonary disease (COPD). However, the molecular mechanisms involved in Cd-induced pulmonary diseases are poorly understood. Here, we identified a cluster of genes that are altered in response to Cd exposure in human bronchial epithelial cells (BEAS-2B) and demonstrated that Cd-induced ER stress and inflammation are mediated via CCAAT-enhancer-binding proteins (C/EBP)-DNA-damaged-inducible transcript 3 (DDIT3) signaling in BEAS-2B cells. Cd treatment led to marked upregulation and downregulation of genes associated with the cell cycle, apoptosis, oxidative stress and inflammation as well as various signal transduction pathways. Gene set enrichment analysis revealed that Cd treatment stimulated the C/EBP signaling pathway and induced transcriptional activation of its downstream target genes, including DDIT3. Suppression of DDIT3 expression using specific small interfering RNA effectively alleviated Cd-induced ER stress and inflammatory responses in both BEAS-2B and normal primary normal human bronchial epithelial cells. Taken together, these data suggest that C/EBP signaling may have a pivotal role in the early induction of ER stress and inflammatory responses by Cd exposure and could be a molecular target for Cd-induced pulmonary disease.

세포투과단백질을 이용한 CARM1 단백질의 직접도입으로 인간 중간엽 줄기 세포에서 나타나는 분화관련 특이 유전자 발현의 변화

조중현,송행석,이동률 한국발생생물학회 2011 한국발생생물학회 학술발표대회 Vol.30 No.-

Protein arginine methyltransferase (PRMT) family 단백질은 히스톤 H3의 arginine 잔기를 메틸레이션 시켜줌으로써 chromatin remodeling을 유발하여 다양한 유전자의 전사를 활성화시켜 주는 것으로 알려져 있다. Coactivator-associated arginine methyltransferase 1 (CARM1/PRMT4)이 그 중의 하나로, 이는 발생과정에서 폐세포로의 분화에 관여하는 것으로 알려져 있으며, 세포의 종류에 따라서 지방세포와 근육세포로의 분화에 영향을 줄 뿐만 아니라, 생쥐 배아줄기세포의 전분화능 유지에도 관여한다는 연구 보고가 있다. 그러나 CARM1이 인간 중간엽 줄기 세포 (hMSCs)에 어떤 영향을 미치는지에 대해서는 현재까지 알려진 바가 없다. 따라서 본 연구에서는 인간 중간엽 줄기세포에 CARM1 단백질을 직접 도입함으로써 변화되는 유전자의 발현 양상을 관찰하였다. 먼저, CARM1 단백질에 세포침투단백질 (cell-penetrating peptide) 서열을 접합시킴으로써, CARM1 단백질이 효율적으로 세포 내로 도입되도록 하였으며, 그 결과 CARM1 단백질이 6시간 내에 충분히 세포막을 통과하여 세포질 및 핵 내로 이동하며, 24시간 내에는 대부분의 단백질이 핵 내에 존재하는 것을 관찰하였다. 또한 도입된 CARM1 단백질이 히스톤 H3의 arginine 잔기를 메틸화시키는 것을 확인하였다. 이렇게 순수분리 정제된 재조합 CARM1 단백질의 처리 후, chromatin remodeling이 일어난 인간 중간엽 줄기세포의 유전자 발현 변화 양상을 microarray를 통해 확인한 결과, 지방세포, 골세포, 근육세포, 신경세포, 그리고 췌장세포 분화에 관여하는 전체 유전자의 약 35~45%의 유전자에서 발현양상의 변화가 나타났다. 따라서 본 연구는 재조합 CARM1 단백질의 세포 내 직접 도입이 CARM1 유전자의 도입으로 우려되는 문제들을 보완하면서도 CARM1 단백질 본연의 기능을 효과적으로 수행한다는 것을 보여주며, 생체 친화적 단백질에 의한 인간 중간엽 줄기세포의 유전자 발현 양상의 변화를 통해 그의 분화와 관련한 임상적 이용의 가능성을 보다 높일 수 있다는 점을 시사한다.

신생 생쥐 고환에서 기인한 다분화능 생식줄기세포주의 확립 및 특성 분석

한상철,송행석,전진현,Han, Sang-Chul,Song, Haeng-Seok,Jun, Jin-Hyun 대한생식의학회 2008 Clinical and Experimental Reproductive Medicine Vol.35 No.1

목 적: 본 연구에서는 신생 생쥐 고환으로부터 다분화능 생식줄기세포주 (MGSCs)를 확립하고, 배아체 형성을 통한 삼배엽성 세포로의 분화 가능성을 확인하고자 하였다. 연구방법: 고환에서 유래한 MGSCs를 확립하기 위하여 생후 $2{\sim}3$일된 생쥐 고환 조직으로부터 세포들을 분리하여 1% FBS를 첨가한 생쥐 배아줄기세포주 배양조건에서 배양하였다. MGSCs 콜로니가 형성된 후에는 배양액의 FBS의 농도를 15%로 높였다. 이러한 과정으로 확립된 MGSCs의 미분화 및 분화 특성을 배아줄기세포주와 비교, 분석하였다. 결 과: 신생 생쥐 고환 조직에서 수획한 세포들로 실시한 9번의 배양실험에서 2개의 MGSCs 세포주를 확립하였다. MGSCs 세포주와 생쥐 배아줄기세포 모두에서 미분화 표지인자인 Thy-1, Oct-4, Nanog, Sox2의 발현과 alkaline phosphatase 활성을 관찰할 수 있었으며, MGSCs의 미세구조 또한 생쥐 배아줄기세포와 유사하였다. MGSCs에서 형성된 배아체에서 삼배엽성 표지유전자의 발현을 확인하였다. 결 론: 본 연구의 결과는 배아줄기세포의 윤리적인 문제점을 극복할 수 있는 고환 유래의 다분화능 MGSCs가 생물공학과 재생의학에서 효율적으로 이용될 수 있는 가능성을 보여준 것으로 생각된다. Objective: The aim of this study was to investigate whether multipotent germline stem cells (MGSCs) can be established from neonatal mouse testis. Methods: Various cells containing MGSCs were collected from neonatal testis of ICR mice and allocated to plates for in vitro culture. After 7 days in culture, the cells were passed to a fresh culture plate and continuously cultured. From the third or fourth passage, the presumed MGSCs were cultured and maintained on mitomycin C-inactivated STO feeder cells. The MGSCs were cultured in a condition where mouse embryonic stem cells (ESCs) are cultured. Characteristics of the MGSCs were evaluated by RT-PCR, immunocytochemistry, alkaline phosphatase activity, karyotyping, and transmission electron microscopy. Results: Two MGSCs lines were established from 9 pooled sets of neonatal testicular cells. MGSCs colonies were morphologically undistinguishable from ESCs colonies and both MGSC lines as well as ESCs expressed undifferentiated stem cell markers, such as Thy-1, Oct-4, Nanog, Sox2 and alkaline phosphatase. Fine structure of undifferentiated MGSCs were similar to those of ESCs and 60% of MGSCs (12/20) had normal karyotype at passage 10. They were able to form embryoid bodies (EBs) and MGSC-derived EBs expressed marker genes of three germ layers. Conclusion: We could establish the MGSCs from neonatal mouse testis and they were differentiated to multipotent lineages of three germ layers. Molecular characteristics of MGSCs were similar to those of ESCs. Our results suggest a possibility that multipotent stem cells derived from testis, the MGSCs, could replace the ESCs in biotechnology and regenerative medicine.

P-152 Cadmium-induced ER stress and inflammation are mediated through C/EBP-DDIT3 signaling in human bronchial epithelial cells

허혜련,김지영,송행석,김정운,김혜련,홍윤기,양세란,한선숙,이승준,김우진,홍석호 대한결핵 및 호흡기학회 2017 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.124 No.0

Cadmium (Cd), a major component of cigarette smoke, disrupts the normal functions of airway cells and can lead to the development of various pulmonary diseases such as chronic obstructive pulmonary disease (COPD). However, the molecular mechanisms involved in Cd-induced pulmonary diseases are poorly understood. Here, we identified a cluster of genes that are altered in response to Cd exposure in human bronchial epithelial cells (BEAS-2B) and demonstrated that Cd-induced ER stress and inflammation are mediated via CCAAT-enhancer-binding proteins (C/EBP)-DNA-damaged-inducible transcript 3 (DDIT3) signaling in BEAS-2B cells. Cd treatment led to marked upregulation and downregulation of genes associated with the cell cycle, apoptosis, oxidative stress and inflammation as well as various signal transduction pathways. Gene set enrichment analysis revealed that Cd treatment stimulated the C/EBP signaling pathway and induced transcriptional activation of its downstream target genes, including DDIT3. Suppression of DDIT3 expression using specific small interfering RNA effectively alleviated Cd-induced ER stress and inflammatory responses in both BEAS-2B and normal primary normal human bronchial epithelial cells. Taken together, these data suggest that C/EBP signaling may have a pivotal role in the early induction of ER stress and inflammatory responses by Cd exposure and could be a molecular target for Cd-induced pulmonary disease. This work was supported by NRF(National Research Foundation of Korea) Grant funded by Korean Government (2016H1A2A1909769-Global Ph.D. Fellowship program)

Gestational Diabetes Affects the Growth and Functions of Perivascular Stem Cells

안보림,김은비,송행석,하권수,한은택,박원선,안태규,양세란,나성훈,홍석호 한국분자세포생물학회 2017 Molecules and cells Vol.40 No.6

Gestational diabetes mellitus (GDM), one of the common metabolic disorders of pregnancy, leads to functional alterations in various cells including stem cells as well as some abnormalities in fetal development. Perivascular stem cells (PVCs) have gained more attention in recent years, for the treatment of various diseases. However, the effect of GDM on PVC function has not been investigated. In our study, we isolated PVCs from umbilical cord of normal pregnant women and GDM patients and compared their phenotypes and function. There is no significant difference in phenotypic expression, response to bFGF exposure and adipogenic differentiation capacity between normal (N)-PVCs and GDM-PVCs. However, when compared with N-PVCs, early passage GDM-PVCs displayed decreased initial rates of cell yield and prolifer-ation as well as a reduced ability to promote wound closure. These results suggest that maternal metabolic dysregulation during gestation can alter the function of endogenous multipotent stem cells, which may impact their therapeutic effectiveness.

Expression of Egr3 in mouse gonads and its localization and function in oocytes

신혜진,설동원,남민영,송행석,이동율,임현정 아세아·태평양축산학회 2017 Animal Bioscience Vol.30 No.6

Objective: The early growth response (Egr) family consists of four members (Egr1, Egr2, Egr3, and Egr4) that are zinc finger transcription factors. Among them, Egr3 is involved in transcriptional regulation of target genes during muscle spindle formation and neurite outgrowth. We previously showed that the immunoreactive Egr3 is localized on oocyte spindle and accumulate near the microtubule organizing center during meiosis I in mice. Egr3 was also shown to be localized on spermatocytes. We herein investigated if Egr3 is expressed in mouse gonads and if Egr3 blockade results in any defect in oocyte maturation. Methods: Expression of Egr3 in mouse gonads was examined by reverse transcription-polymerase chain reaction. Full-length Egr3 and truncated Egr3 (ΔEgr3) complementary RNAs (cRNAs) with Xpress tag at N-terminus and DsRed2 at C-terminus, and small interfering RNA (siRNA) targeting Egr3 were microinjected into mouse oocytes at germinal vesicle stage. Localization of microinjected Egr3 was examined by confocal live imaging and immunofluorescence staining. Results: Egr3 mRNA was detected in mouse ovaries and testes from 1 to 4 week-old mice. An uncharacterized longer transcript containing 5’untranslated region was also detected in 3 and 4 week-old gonads. Microinjected Xpress-Egr3-DsRed2 or Xpress-ΔEgr3-DsRed2 localized to nuclei and chromosomes during meiotic progression. Microinjection of these cRNAs or Egr3 siRNA in oocytes did not affect meiotic maturation. Immunofluorescence staining of Egr3 in Xpress-ΔEgr3-DsRed2-injected oocytes showed a positive signal only on meiotic spindle, suggesting that this antibody does not detect endogenous or exogenous Egr3 in mouse oocytes. Conclusion: The results show that Egr3 localizes to chromosomes during meiotic progression and that certain antibodies may not faithfully represent localization of target proteins in oocytes. Egr3 seems to be dispensable during oocyte maturation in mice.

Identification of Egr1 Direct Target Genes in the Uterus by In Silico Analyses with Expression Profiles from mRNA Microarray Data

서봉정,손지원,김혜련,홍석호,송행석 한국발생생물학회 2014 발생과 생식 Vol.18 No.1

Early growth response 1 (Egr1) is a zinc-finger transcription factor to direct second-wave gene expression leading to cell growth, differentiation and/or apoptosis. While it is well-known that Egr1 controls transcription of an array of targets in various cell types, downstream target gene(s) whose transcription is regulated by Egr1 in the uterus has not been identified yet. Thus, we have tried to identify a list of potential target genes of Egr1 in the uterus by performing multi-step in silico promoter analyses. Analyses of mRNA microarray data provided a cohort of genes (102 genes) which were differentially expressed (DEGs) in the uterus between Egr1(+/+) and Egr1(–/–) mice. In mice, the frequency of putative EGR1 binding sites (EBS) in the promoter of DEGs is significantly higher than that of randomly selected non-DEGs, although it is not correlated with expression levels of DEGs. Furthermore, EBS are considerably enriched within –500 bp of DEG’s promoters. Comparative analyses for EBS of DEGs with the promoters of other species provided power to distinguish DEGs with higher probability as EGR1 direct target genes. Eleven EBS in the promoters of 9 genes among analyzed DEGs are conserved between various species including human. In conclusion, this study provides evidence that analyses of mRNA expression profiles followed by two-step in silico analyses could provide a list of putative Egr1 direct target genes in the uterus where any known direct target genes are yet reported for further functional studies.

과립구대식세포 증식인자가 사람 영양막세포의 유전자 발현에 미치는 영향

이헌열 ( Hun Yul Lee ),김상수 ( Sang Soo Kim ),안창숙 ( Chang Suk Ahn ),박영주 ( Young Joo Park ),전진현 ( Jin Hyun Jun ),송행석 ( Haeng Suk Song ),문혜성 ( Hye Sung Moon ),박은주 ( Eun Joo Park ),홍서유 ( Seo You Hong ),박원일 ( 대한산부인과학회 2010 Obstetrics & Gynecology Science Vol.53 No.10

목적: 포유동물의 생식과정에서 Granulocyte-macrophage colony-stimulating factor (GM-CSF)가 배아의 침윤성을 증가시킨다는 사실은 알려졌으나 착상 이후와 태반형성에서의 작용은 알려진 바가 거의 없다. 본 연구는 GM-CSF가 임신초기 사람의 영양막세포에서도 침윤성을 증가시키는 작용이 있는 지를 확인하는 것이 목적이다. 연구 방법: 임신초기 소파술을 통해 얻은 사람의 영양막세포를 배양한 후 GM-CSF 처리군과 대조군의 유전자 발현 차이를 microarray를 통하여 비교하였고 차이가 있는 유전자 중 착상과 관련된 유전자의 발현을 real-time RT-PCR로 확인하였다. 결과: Microarray 결과 GM-CSF 처리군에서 2배 이상 증가한 유전자는 468개, 2배 이상 감소한 유전자는 40개였다. 이 유전자를 생물학적 pathway에 따라 분석한 결과 actin cytoskeleton 및 focal adhesion에 관여된 유전자가 많았다. Actin cytoskeleton 및 focal adhesion에 관여된 유전자 중 annexin A2, thymosin-like 3, vimentin, myogenin, ACK1, tensin1의 발현을 real-time RT-PCR로 확인하였다. 그 결과 GM-CSF에 의해 vimentin과 ACK1의 발현은 증가되었고 tensin1의 발현은 감소되었다. 결론: GM-SF는 사람의 초기 임신 영양막세포에서 vimentin과 ACK1의 발현을 증가시키고 tensin1을 감소시킴으로써 focal adhesion을 증가시킨다고 생각된다. Objective: Granulocyte-macrophage colony-stimulating factor (GM-CSF) facilitates mammalian embryonic development and implantation. However, its biological function after implantation is not elucidated. The aim of this study is to assess the changes of gene expression by GM-CSF in human trophoblast obtained in early pregnancy. Methods: Human trophoblast obtained in early pregnancy was cultured with or without GM-CSF. The difference of gene expression was evaluated with microarray and selected genes were reevaluated with real-time reverse transcription-polymerase chain reaction (RT-PCR). Results: Microarray analysis revealed that the expressions of 468 genes were increased while those of 40 genes were decreased by GM-CSF. These genes were evaluated according to the known biologic pathways. The regulation of actin cytoskeleton and focal adhesion pathways were mostly influenced by GM-CSF. Annexin A2, thymosin-like 3, vimentin, myogenin, ACK1, and tensin1 genes were selected for real-time RT-PCR. The increased expressions of of vimentin and ACK1, and decreased expressions of tensin1 were confirmed by real-time RT-PCR. Conclusion: GM-CSF activates focal adhesion pathway in human trophoblast by increasing the expression of vimentin and ACK1, and decreasing the expression of tensin1.