Establishment of a Human Embryonic Germ Cell Line and Comparison with Mouse and Human Embryonic Stem Cells

Hyun Soo Yoon,김철근,Jong Hyuk Park,Sun Jong Kim,Jung Bok Lee,Ji Min Song,Sung Il Roh 한국분자세포생물학회 2004 Molecules and cells Vol.17 No.2

Human embryonic stem (ES) cells and embryonic germ (EG) cells are pluripotent and are invaluable material for in vitro studies of human embryogenesis and cell therapy. So far, only two groups have reported the establishment of human EG cell lines, whereas at least five human ES cell lines have been established. To see if human EG cell lines can be reproducibly estab-lished, we isolated primordial germ cells (PGCs) from gonadal ridges and mesenteries (9 weeks post-fertilization) and cultured them on mouse STO cells. As with mouse ES colonies, the PGC-derived cells have given rise to multilayered colonies without any differ-entiation over a year of continuous culture. They are karyotypically normal and express high levels of alka-line phosphatase, Oct-4, and several cell-surface mark-ers. Histological and immunocytochemical analysis of embryoid bodies (EBs) formed from floating cultures of the PGC-derived cell colonies revealed ectodermal, endodermal, and mesodermal tissues. When the EBs were cultured in the presence of insulin, transferrin, sodium selenite, and fibronectin for 1 week, markers of primitive neuroectoderm were expressed in cells within the EBs as well as in cells growing out from the EBs. These observations indicate that our PGC-derived cells satisfy the criteria for pluripotent stem cells and hence may be EG cells.

NMDA Receptor as a Newly Identified Member of the Metabotropic Glutamate Receptor Family: Clinical Implications for Neurodegenerative Diseases

정지혜 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.2

Recent reports have proposed a novel function for the N-methyl-D-aspartate (NMDA) receptor (NMDAR), a well-known excitatory, ionotropic receptor. A series of observations employing pharmacological techniques has proposed that upon ligand binding, this ionotropic receptor can actually function via signaling cascades independent of traditional ionotropic action. Moreover, the “metabotropic” action of NMDARs is suggested to mediate a form of synaptic plasticity, namely long-term synaptic depression (LTD), which shares cellular mechanisms with the synaptic deficits observed in Alzheimer’s disease. Given that a growing body of clinical and preclinical evidence strongly recommends NMDAR antagonists for their therapeutic potentials and advantages in a variety of diseases, further investigation into their molecular and cellular mechanisms is required to better understand the “metabotropic” action of NMDARs.

TrkC Promotes Survival and Growth of Leukemia Cells Through Akt-mTOR-Dependent Up-Regulation of PLK-1 and Twist-1

김민수,진욱,김경미,최윤정,김혜정,김유진 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.2

It has been suggested that activation of receptor PTKs is important for leukemogenesis and leukemia cell response to targeted therapy in hematological malignancies including leukemia. PTKs induce activation of the PI3K/Akt/mTOR pathway, which can result in prevention of apoptosis. Here, we describe an important role of the TrkC-associated molecular network in the process of leukemogenesis. TrkC was found to be frequently overexpressed in human leukemia cells and leukemia subtypes. In U937 human leukemia cells, blockade of TrkC using small hairpin RNA (shRNA) specific to TrkC or K562a, a specific inhibitor of TrkC, resulted in a significant decrease in growth and survival of the cells, which was closely associated with reduced mTOR level and Akt activity. In addition, TrkC enhances the survival and proliferation of leukemia, which is correlated with activation of the PI3K/Akt pathway. Moreover, TrkC significantly inhibits apoptosis via induction of the expression of PLK-1 and Twist-1 through activation of AKT/mTor pathway; therefore, it plays a key role in leukemogenesis. These findings reveal an unexpected physiological role for TrkC in the pathogenesis of leukemia and have important implications for understanding various hematological malignancies.

Establishment of a Bone-Specific col10a1:GFP Transgenic Zebrafish

김용일,김철희,이수만,정성현,김현택,최정화,이미선,유관희,여상엽,유경원,곽성애,이준노,박래길,최성규 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.2

During skeletal development, both osteogenic and chondrogenic programs are initiated from multipotent mesenchymal cells, requiring a number of signaling molecules, transcription factors, and downstream effectors to orchestrate the sophisticated process. Col10a1, an important downstream effector gene, has been identified as a marker for maturing chondrocytes in higher vertebrates, such as mammals and birds. In zebrafish, this gene has been shown to be expressed in both osteoblasts and chondrocytes, but no study has reported its role in osteoblast development. To initially delineate the osteogenic program from chondrogenic lineage development, we used the zebrafish col10a1 promoter to establish a transgenic zebrafish expressing a GFP reporter specifically in osteoblast-specific bone structures that do not involve cartilaginous programs. A construct harboring a ~2.2-kb promoter region was found to be sufficient to drive the reporter gene in osteoblast-specific bone structures within the endoge-nous col10a1 expression domain, confirming that separable cis-acting elements exist for distinct cell type-specific expression of col10a1 during zebrafish skeletal development. The ~2.2-kb col10a1:GFP trans- genic zebrafish marking only bone structures derived from osteoblasts will undoubtedly be an invaluable tool for identifying and characterizing molecular events driving osteoblast development in zebrafish, which may further provide a differential mechanism where col10a1 is involved in the development of chondrocytes undergoing maturation in other vertebrate systems.

Tbc1d15-17 Regulates Synaptic Development at the Drosophila Neuromuscular Junction

이민정,이승복,장수연,남민엽,윤진호 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.2

Members of the Tre-2/Bub2/Cdc16 (TBC) family of pro-teins are believed to function as GTPase-activating pro-teins (GAPs) for Rab GTPases, which play pivotal roles in intracellular membrane trafficking. Although membrane trafficking is fundamental to neuronal morphogenesis and function, the roles of TBC-family Rab GAPs have been poorly characterized in the nervous system. In this paper, we provide genetic evidence that Tbc1d15-17, the Drosophila homolog of mammalian Rab7-GAP TBC1d15, is required for normal presynaptic growth and postsynaptic organization at the neuromuscular junction (NMJ). A loss-of-function mutation in tbc1d15-17 or its presynaptic knock- down leads to an increase in synaptic bouton number and NMJ length. tbc1d15-17 mutants are also defective in the distribution of the postsynaptic scaffold Discs-large (Dlg) and in the level of the postsynaptic glutamate subunit GluRIIA. These postsynaptic phenotypes are recapitulated by postsynaptic knockdown of Tbc1d15-17. We also show that presynaptic overexpression of a constitutively active Rab7 mutant in a wild-type background causes a synaptic overgrowth phenotype resembling that of tbc1d15-17 mutants, while a dominant-negative form of Rab7 has the opposite effect. Together, our findings establish a novel role for Tbc1d15-17 and its potential substrate Rab7 in regulating synaptic development.

Wif1 Hypermethylation as Unfavorable Prognosis of Non-Small Cell Lung Cancers with EGFR Mutation

이수만,김동선,박재용 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.1

Lung cancer is a leading cause of cancer-related mortal-ity across the world and tobacco smoking is the major risk factor. The Wnt signaling pathway is known to be involved in smoke-induced tumorigenesis in the lung. Promoter hypermethylation of Wnt inhibitory factor 1 (Wif1) has become a common event in a number of human tumors. Using a methylation-specific PCR, hypermethylation of the Wif1 gene promoter was evaluated in 139 primary non-small cell lung cancers (NSCLCs) and its correlation with clinicopathological and prognostic parameters was evaluated. Methylation of Wif1 was observed in 47.5% and 20.9% of neoplastic and adjacent normal lung tissues, respectively. Its methyla-tion rate tended to be higher in stage I than stages II-IIIA. Results of Kaplan-Meier analysis showed no significant difference in overall survival accor-ding to Wif1 methylation status. However, Wif1 methylation showed an association with unfavorable prognosis of ad-enocarcinoma (AC) patients with EGFR mutation. According to our current findings, Wif1 promoter methylation is an early, frequent event as an epigenetic field manner and could be considered as a useful prognostic marker for AC patients with EGFR mutation. Further investigation into the therapeutic potential of this finding is warranted.

Regulation of Transcription from Two ssrS Promoters in 6S RNA Biogenesis

이지영,이영훈,박홍만,백근우,김광선 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.3

ssrS-encoded 6S RNA is an abundant noncoding RNA that binds σ70-RNA polymerase and regulates expression at a subset of promoters in Escherichia coli. It is tran-scribed from two tandem promoters, ssrS P1 and ssrS P2. Regulation of transcription from two ssrS promoters in 6S RNA biogenesis was examined. Both P1 and P2 were growth phase-dependently regulated. Depletion of 6S RNA had no effect on growth-phase-dependent transcription from either promoter, whereas overexpression of 6S RNA increased P1 transcription and decreased P2 transcription, suggesting that transcription from P1 and P2 is subject to feedback activation and feedback inhibition, respectively. This feedback regulation disappeared in Δfis strains, supporting involvement of Fis in this process. The differential feedback regulation may provide a means for maintaining appropriate cellular concentrations of 6S RNA.

Sphingosine and FTY720 Modulate Pacemaking Activity in Interstitial Cells of Cajal from Mouse Small Intestine

남주현,김병주,김우경 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.3

Interstitial cells of Cajal (ICCs) are the pacemakers of the gastrointestinal tract, and transient receptor potential melastatin type 7 (TRPM7) and Ca2+ activated Cl- channels (ANO1) are candidate the generators of pacemaker potentials in ICCs. The effects of D-erythro-sphingosine (SPH) and structural analogues of SPH, that is, N,N-dimethyl-D-erythro-sphingosine (N,N-DMS), FTY720, and FTY720-P on the pacemaking activities of ICCs were examined using the whole cell patch clamp technique. SPH, N,N-DMS, and FTY720 decreased the amplitudes of pacemaker potentials in ICC clusters, but resting membrane potentials displayed little change. Also, perfusing SPH, N,N-DMS, or FTY720 in the bath reduced both inward and outward TRPM7-like currents in single ICCs, and inhibited ANO1 currents. The another structural analogue of SPH, FTY720-P was ineffective at the pacemaker potentials in ICC clusters and the TRPM7-like currents in single ICCs. Furthermore, FTY720-P had no effect on ANO1. These results suggest that SPH, N,N-DMS, and FTY720 modulate the pacemaker activities of ICCs, and that TRPM7 and ANO1 channels affect intes-tinal motility.

Leucine-Rich Repeat-Containing G-Protein Coupled Receptor 5/GPR49 Activates G12/13-Rho GTPase Pathway

권미소,김선홍,박비오,김호민 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.3

Leucine-rich repeat-containing G-protein coupled recep-tor 5 (LGR5/GPR49) is highly expressed in adult stem cells of various tissues, such as intestine, hair follicles, and stomach. LGR5 is also overexpressed in some colon and ovarian tumors. Recent reports show that R-spondin (RSPO) family ligands bind to and activate LGR5, enhancing canonical Wnt signaling via the interaction with LRP5/6 and Frizzled. The identity of heterotrimeric G-proteins coupled to LGR5, however, remains unclear. Here, we show that Rho GTPase is a downstream target of LGR5. Overexpression of LGR5 induced SRF-RE luciferase activity, a reporter of Rho signaling. RSPOs, ligands for LGR4, LGR5, and LGR6, however, did not induce SRF-RE reporter activity in the presence of LGR5. Consistently, LGR5-induced activity of the SRF-RE reporter was inhibited by Rho inhibitor C3 transferase and RhoA N19 mutant, and knockdown of G12/13 genes blocked the reporter activity induced by LGR5. In addition, focal adhesion kinase, NF-B and c-fos, targets of Rho GTPase, were shown to be regulated by LGR5. Here, we have demonstrated, for the first time, that LGR5 is coupled to the Rho pathway through G12/13 signaling.

Selective Induction of P2Y14 Receptor by RANKL Promotes Osteoclast Formation

이승아,이수영,박진희 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.3

The purinergic receptor P2Y, G protein coupled, 14 (P2Y14) receptor for UDP-glucose and other UDP-sugars has been implicated in the regulation of the stem cell compartment as well as neuroimmune function. However, the role of P2Y14 in osteoclast formation is completely unknown. We found that RANKL selectively induced P2Y14 among seven mammalian P2Y receptors when analysed at both the mRNA and protein level, but inhibitors of the mitogen-activated protein (MAP) kinase pathway suppressed induction of P2Y14 proteins. Extracellular addition of UDP-su-gars such as UDP-glucose, UDP-galactose, UDP-glucuro-nic acid, and UDP-N-acetyl glucosamine promoted RANKL- induced osteoclastogenesis, while P2Y14 downregulation by RNA interference inhibited osteoclast formation. Taken together, these results suggest that P2Y14 may act as the receptor for UDP-sugars in osteoclast precusors and may regulate RANKL-induced osteoclastogenesis.