• Journal of Life Science
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• v.25 no.2
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• pp.223-230
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• 2015

Regulator of calcineurin 1 (RCAN1) is an endogenous calcineurin inhibitor that plays an important role in the pathogenesis of diseases related to the calcineurin-NFATc1 signaling pathway. The RCAN1-4 isoform is subject to NFATc1-dependent regulation. During receptor activator of nuclear factor kappa-B ligand (RANKL)-stimulated osteoclastogenesis, the calcineurin-NFATc1 pathway is critical. Because there is little information available on the role of RCAN1 in osteoclast differentiation, this study investigated whether changes in RCAN1 expression are related to the calcineurin-NFATc1 pathway and osteoclast differentiation. Mouse bone marrow monocytes (BMMs) were treated with 50 ng/ml of RANKL and M-CSF. Expression levels of NFATc1, calcineurin, and RCAN1 isoforms were determined using RT-PCR and Western blotting. Osteoclast differentiation was examined using tartrate-resistent acid phosphatase (TRAP) staining. To evaluate the effect of RCAN1 overexpression on osteoclastogenesis, cells were transfected with a mouse RCAN1-4 cDNA plasmid. After RANKL stimulation of BMMs, expression of NFATc1 and RCAN1 was increased at the mRNA and protein level, while calcineurin expression was unchanged. When the RCAN1-4 gene construct was transfected, the expression of RCAN1 protein was not increased despite several-fold increases in RCAN1-4 mRNA expression. Regardless of RANKL stimulation, over-expression of RCAN1-4 tended to reduce NFATc1 expression and knock-down of RCAN1 increase it. While BMMs transfected with the RCAN1-4 vector were differentiated into distinct osteoclasts, their phenotypes did not vary from those of mock controls. These results suggest that RCAN1 has a limited effect on the calcineurin-NFATc1 pathway during RANKL-stimulated osteoclast differentiation.

• Development and Reproduction
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• v.11 no.3
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• pp.235-243
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• 2007

Human umbilical cord blood(HUCB) contains a rich source of hematopoietic stem cells, mesenchymal stem cells and endothelial cell precursors. Mesenchymal stem cells(MSCs) in HUCB are multipotent stem cells, differ from hematopoietic stem cells and can be differentiated into neural cells. We studied on transdifferentiation-promoting conditions in neural cells and cholinergic neuron induction of HUCB-derived MSCs. Neural differentiation was induced by addingdimethyl sulphoxide(DMSO) and butylated hydroxyanisole(BHA) in Dulbeco's Modified Essential Medium(DMEM) and fetal bovine serum(FBS). Differentiation of MSCs to cholinergic neurons was induced by combined treatment with basic fibroblast growth factor(bFGF), retinoic acid(RA) and sonic hedgehog(Shh). MSCs treated with DMSO and BHA rapidly assumed the morphology of multipolar neurons. Both immunocytochemistry and RT-PCR analysis indicated that the expression of a number of neural markers including $\beta$-tubulin III, GFAP and MBP, was markedly elevated during this acute differentiation. The differentiation rate was about $32.3{\pm}2.9%$ for $\beta$-tubulin III-positive cells, $11.0{\pm}0.9%$ for GFAP, and $9.4{\pm}1.0%$ for Gal-C. HUCB-MSCs treated combinatorially with bFGF, RA and Shh were differentiated into cholinergic neurons. After cholinergic neuronal differentiation, the $\beta$-tubulin III-positive cell population of total cells was $31.3{\pm}3.2%$ and of differentiated neuronal population, $70.0{\pm}7.8%$ was ChAT-positive showing 3 folds higher in cholinergic population than neural induction. Conclusively, HUCB-derived MSCs can be differentiated into neural and cholinergic neurons and these findings suggest that HUCB are alternative cell source of treatment for neurodegenerative diseases such as Alzheimer's disease.

• Journal of Life Science
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• v.28 no.12
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• pp.1397-1405
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• 2018

Mitochondria have multiple functions in cells: providing chemical energy, storing cellular $Ca^{2+}$, generating reactive oxygen species, and regulating apoptosis. Through these functions, mitochondria are also involved in the maintenance, proliferation, and differentiation of stem/progenitor cells. In the brain, the subventricular zone (SVZ) is one of the neurogenic regions that contains neural stem cells (NSCs) throughout a lifetime. However, reports on the role of mitochondria in SVZ NSCs are scarce. Here, we show that rotenone, a complex I inhibitor of mitochondria, inhibits the proliferation and differentiation of SVZ NSCs in different ways. In proliferating NSCs, rotenone decreases mitosis as measured through phosphorylated histone H3 detection; moreover, apoptosis is not induced by rotenone at 50 nM. In differentiating NSCs, rotenone blocks neurogenesis and oligodendrogenesis while glial fibrillary acidic protein-positive astrocytes are not affected. Interestingly, in this study there were more cells in the differentiating NSCs treated with rotenone for 4-6 days than in the vehicle control group which was a different effect from the reduced number of cells in the proliferating NSCs. We examined both apoptosis and mitosis and found that rotenone decreased apoptosis as detected by staining cleaved caspase-3 but did not affect mitosis. Our results suggest that functional mitochondria are necessary in both the proliferation and differentiation of SVZ NSCs. Furthermore, mitochondria might be involved in the mitosis and apoptosis that occur during those processes.

• Korean Journal of Plant Tissue Culture
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• v.22 no.3
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• pp.169-173
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• 1995

Border cells are differentiated cells which originate from meristematic cells in The root cap. Experimentally border cells can be released from the root cap by a physical treatment, for example dipping the root tip in the waters After 20-25 hours of release, the new border cell layer forms in the root cap. During the border cell differentiation, new gene expressions were observed in the root cap of pea which was determined by mRNA differential display These new gene expressions may be involved in the border cell differentiation Border cells had unique gene expressions which were determined by mRNA differential display, This suggests that border cells are differentiated cells which are different from the other tissues (ie., leaves, stems, roots or root caps).

• Proceedings of the Plant Resources Society of Korea Conference
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• 2018.04a
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• pp.79-79
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• 2018

현대사회에서 스트레스, 환경오염 등으로 인하여 중년남성의 전유물로 여겨져 왔던 탈모가 젊은 사람들에게서도 나타나며, 탈모로 고민하는 인구가 약 700만 명 이상으로 추산되고 계속해서 증가하는 추세이다. 우리나라 탈모관련 시장은 현재 약 4조원 규모이며 지속적인 성장세에 있으며, 주로 한약재등 천연추출물을 혼합하여 제품화하였다. 본 연구에서는 어성초, 금잔화 70% 에탄올 추출물을 이용하여 천연발모제 소재화를 목적으로 섬유아세포, 모유두세포, 지방전구세포에 대한 세포독성 및 지방전구세포 분화를 통한 발모 효능을 확인하여 약용작물을 이용한 향장품 소재화를 시도하고자 한다. 섬유아세포에 각 시료에 대한 세포 독성을 확인한 결과는 어성초 70% 에탄올 추출물을 50, 100, $500{\mu}g/mL$으로 처리하였을 때 107.3%, 109.6%, 128.2%로 세포 생존율이 증가하였다. 모유두세포에 대한 각 시료의 세포 독성은 나타나지 않았으며, 모발의 생육을 촉진하는 혈소판에서 유래하는 성장인자를 분비하여 모발 재생에 효과를 나타내는 지방전구 세포의 지방 분화 확인 결과 금잔화와 어성초 70% 에탄올 추출물을 $10{\mu}g/mL$의 농도로 처리하였을 때 각각 104.3%, 103.9%의 분화율을 나타내었고, 어성초 출물의 경우 처리 농도의 증가에 따라 지방분화율이 유의적으로 크게 증가하였다. 본 연구를 통해 육모용 모발제품 소재 개발을 위한 생약초 자원들의 in-vitro 육모활성관련 실험 결과 어성초와 금잔화는 각 세포에 대한 독성을 나타내지 않았으며, 어성초 70% 에탄올 추출물은 지방전구세포를 자극하여 지방 분화를 유도하는 것으로 확인되었다. 이상과 같은 결과로 어성초와 금잔화 70% 에탄올 추출물은 탈모관련 향장품 개발 소재로 높은 가능성을 가지고 있는 것으로 생각된다.

• Journal of Life Science
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• v.30 no.12
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• pp.1092-1100
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• 2020

The six-transmembrane epithelial antigen of prostate 4 (Steap4) is a metalloreductase that plays a role in intracellular iron and cupper homeostasis, inflammatory response, and glucose and lipid metabolism. Previously, Steap4 has been reported to stimulate adipocyte differentiation; however, the underlying mechanisms of this action remain unexplored. In the present study, we investigated the molecular mechanisms involved in Steap4-induced adipocyte differentiation using 3T3-L1 cells, immortalized brown adipocyte (iBA) cells, and mouse embryonic fibroblast C3H10T1/2 cells. The knockdown of Steap4 using adenovirus-containing shRNA attenuated mitotic clonal expansion (MCE), as evidenced by the impaired proliferation of 3T3-L1 cells, iBA cells, and C3H10T1/2 cells within 48 hr after adding the differentiation medium. Steap4 knockdown downregulated G1/S phase transition-related cell cycle regulators (including cyclin A and cyclin D) and upregulated cell cycle inhibitors (including p21 and p27). Furthermore, Steap4 knockdown inhibited the phosphorylation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and Akt. Moreover, Steap4 knockdown repressed the expression of early adipogenic activators, such as CCAAT-enhancer-binding protein β (C/EBPβ) and Kruppel-like factor family factor 4 (KLF4). On the other hand, Steap4 knockdown stimulated the expression of adipogenic inhibitors, including KLF2, KLF3, and GATA2. The overexpression of Steap4 using an adenovirus removed the repressive histone marks H3K9me2 and H3K9me3 on the promoter of C/EBPβ. These results indicate that Stepa4 stimulates adipocyte differentiation through the induction of MCE and the modulation of early adipogenic transcription factors, including C/EBPβ, during the early phase of adipocyte differentiation.

• Journal of Life Science
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• v.26 no.1
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• pp.8-16
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• 2016

Reactive oxygen species (ROS), at appropriate concentrations, mediate various normal cellular functions, including defense against pathogens, signal transduction, cellular growth, and gene expression. A recent study demonstrated that ROS and ROS-generating NADPH oxidase (Nox) are important in self-renewal and neuronal differentiation of subventricular zone (SVZ) neural stem cells in adult mouse brains. In this study, we found that endogenous ROS were detected in SVZ neural stem cells cultured from postnatal mouse brains. Nox4 was predominantly expressed in cultured cells, while the levels of the Nox1 and Nox2 transcripts were very low. In addition, the Nox4 gene was highly upregulated (by up to 10-fold) during neuronal differentiation. Immunocytochemical analysis detected the Nox4 protein mainly in neurons positive for the neuronal specific tubulin Tuj1. After differentiation, endogenous ROS were detected exclusively in neuron-like cells with processes. In addition, perturbation of the cellular redox state with N-acetyl cysteine, a ROS scavenger, during neuronal differentiation greatly inhibited neurogenesis. Lastly, knockdown of Nox4 using short hairpin RNA decreased neurogenesis. These findings suggest that Nox4 may be a major ROS-generating enzyme in postnatal SVZ neural stem cells, and Nox4-mediated ROS generation may be important in their neuronal differentiation.

• Journal of Life Science
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• v.23 no.1
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• pp.137-142
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• 2013

Human adipose-derived mesenchymal stem cells (hMSCs) are highly useful for vascular regeneration of injured or inflamed tissue. Lipopolysaccharide (LPS) is a potent activator of macrophages and stimulates macrophages to release inflammatory cytokines. In the present study, we explored the role of LPS-activated macrophages in the differentiation of hMSCs to smooth muscle cells (SMCs). We demonstrated that conditioned medium from LPS-induced macrophages (LPS CM) stimulates differentiation of hMSCs to SMCs, as evidenced by increased expression of smooth muscle-specific markers, including alpha-smooth muscle actin (${\alpha}$-SMA), smooth muscle-myosin heavy chain, and calponin. LPS induced the secretion of $PGF2{\alpha}$ from macrophages, and $PGF2{\alpha}$ treatment stimulated expression levels of SMC-specific markers in hMSCs. Furthermore, small interfering RNA-mediated silencing of the $PGF2{\alpha}$ receptor inhibited LPS CM-stimulated ${\alpha}$-SMA expression. These results suggest that LPS-activated macrophages promote differentiation of hMSCs to SMCs through a $PGF2{\alpha}$-dependent mechanism.

• Microbiology and Biotechnology Letters
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• v.44 no.4
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• pp.557-562
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• 2016

Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis are gram-negative bacteria frequently found in lesions from patients with periodontitis manifesting alveolar bone loss. Lipopolysaccharides are a major virulence factor of gram-negative bacteria. Bone resorption is known to be regulated by bacteria and their virulence factors. In the present study, we investigated the effects of A. actinomycetemcomitans and P. gingivalis on bone resorption. Heat-killed A. actinomycetemcomitans (HKAa) and heatkilled P. gingivalis (HKPg) induced bone loss in the femurs of mice after intraperitoneal administration. HKAa and HKPg augmented the differentiation of committed osteoclast precursors into osteoclasts, while they inhibited the differentiation of bone marrow-derived macrophages into osteoclasts. Concordant with the effects of the heat-killed whole cells, LPS purified from A. actinomycetemcomitans and P. gingivalis also augmented osteoclast differentiation from committed osteoclast precursors but attenuated it from bone marrow-derived macrophages. Taken together, these results suggest that the whole cells and lipopolysaccharides of A. actinomycetemcomitans and P. gingivalis induce the differentiation of committed osteoclast precursors into osteoclasts, potentially contributing to bone resorption in vivo.

• Proceedings of the Materials Research Society of Korea Conference
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• 2010.05a
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• pp.42.1-42.1
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• 2010

Poly-carprolactone (PCL)은 생분해성 고분자로 장기간의 임상실험 결과 생체에 독성이 없으며 생체친화성이 우수한 소재로 확인되어 PLGA, PLLA 등과 더불어 조직공학 분야에서 널리 사용되고 있는 생체재료이다. 그러나 PCL은 5개의 비극성 methylene group과 1개의 극성 ester group이 반복되는 지방족의 polyester로 구조상 탄소수가 많아 소수성을 띄는 단점을 가지고 있다. 이러한 표면이 소수성인 재료의 경우, 초기 단백질 흡착능이 떨어져 세포의 부착이 느린 속도로 일어나므로 세포 분화 및 조직 재생이 더디게 일어난다. 본 연구에서는 소수성의 PCL 표면의 단백질 흡착능을 증가시키기 위해 기능성 amine group을 부착하였으며, 또한 골재생을 촉진시킬 수 있는 세포외 기질인 collagen과 osteopontin을 부착함으로써 고기능성 PCL membrane을 제조하였다. 제조된 PCL membrane은 골재생용 조직공학에의 응용을 위해 지방유래 줄기세포를 이용하여 부착능 및 골세포로의 분화능을 확인하였다. 표면 성질의 변화에 의한 세포의 부착능의 변화를 confocal microscopy을 이용하여 부착에 관여하는 단백질의 발현을 확인하였으며, collagen과 osteopontin에 의한 골세포로의 분화능을 확인하기 위해 real time PCR을 통해 골세포의 분화 표지 유전자의 발현을 비교 분석하였다.