• The Korean Journal of Physiology and Pharmacology
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• 제27권3호
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• pp.277-287
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• 2023

Actin dynamics play an essential role in myogenesis through multiple mechanisms, such as mechanotransduction, cell proliferation, and myogenic differentiation. Twinfilin-1 (TWF1), an actin-depolymerizing protein, is known to be required for the myogenic differentiation of progenitor cells. However, the mechanisms by which they epigenetically regulate TWF1 by microRNAs under muscle wasting conditions related to obesity are almost unknown. Here, we investigated the role of miR-103-3p in TWF1 expression, actin filament modulation, proliferation, and myogenic differentiation of progenitor cells. Palmitic acid, the most abundant saturated fatty acid (SFA) in the diet, reduced TWF1 expression and impeded myogenic differentiation of C2C12 myoblasts, while elevating miR-103-3p levels in myoblasts. Interestingly, miR-103-3p inhibited TWF1 expression by directly targeting its 3'UTR. Furthermore, ectopic expression of miR-103-3p reduced the expression of myogenic factors, i.e., MyoD and MyoG, and subsequently impaired myoblast differentiation. We demonstrated that miR-103-3p induction increased filamentous actin (F-actin) and facilitated the nuclear translocation of Yes-associated protein 1 (YAP1), thereby stimulating cell cycle progression and cell proliferation. Hence, this study suggests that epigenetic suppression of TWF1 by SFA-inducible miR-103-3p impairs myogenesis by enhancing the cell proliferation triggered by F-actin/YAP1.

• Journal of Periodontal and Implant Science
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• 제34권1호
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• pp.205-221
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• 2004

Periodontal ligament(PDL) cells have been known as playing an important roles in periodontal regeneration and gingival fibroblasts are also important to periodontal regeneration by forming connective tissue attachment. There were rare studies about the gene expression patterns of PDL cells and gingival fibroblasts, therefore in this study, we tried cDNA microarray-based gene expression monitoring to explain the functional differences of PDL cells and gingival fibroblasts in vivo and to confirm the characteristics of PDL cells. Total RNA were extracted from PDL cells and gingival fibroblasts of same person and same passages, and mRNA were isolated from the total RNA using Oligotex mRNA midi kit(Qiagen) and then fluorescent cDNA probe were prepared. And microarray hybridization were performed. The gene expression patterns of PDL cells and gingival fibroblasts were quite different. About 400 genes were expressed more highly in the PDL cells than gingival fibroblasts and about 300 genes were more highly expressed in the gingival fibroblasts than PDL cells. Compared growth factor- and growth factor receptor-related gene expression patterns of PDL cells with gingival fibroblasts, IGF-2, IGF-2 associated protein, nerve growth factor, placental bone morphogenic protein, neuron-specific growth- associated protein, FGF receptor, EGF receptor-related gene and PDGF receptor were more highly expressed in the PDL cells than gingival fibroblasts. The results of collagen gene expression patterns showed that collagen type I, type III, type VI and type VII were more highly expressed in the PDL cells than gingival fibroblasts, and in the gingival fibroblasts collagen type V, XII were more highly expressed than PDL cells. The results of osteoblast-related gene expression patterns showed that osteoblast specific cysteine-rich protein were more highly expressed in the PDL cells than gingival fibroblasts. The results of cytoskeletal proteins gene expression patterns showed that a-smooth muscle actin, actin binding protein, smooth muscle myosin heavy chain homolog and myosin light chain were more highly expressed in the PDL cells than gingival fibrobalsts, and ${\beta}-actin$, actin-capping protein(${\beta}$ subunit), actin- related protein Arp3(ARP) and myosin class I(myh-1c) were more highly expressed in the gingival fibroblasts than PDL cells. Osteoprotegerin/osteoclastogenesis inhibitory factor(OPG/OCIF) was more highly expressed in the PDL cells than gingival fibroblasts. According to the results of this study, PDL cells and gingival fibroblasts were quite different gene expression patterns though they are the fibroblast which have similar shape. Therefore PDL cells & gingival fibroblasts are heterogeneous populations which represent distinct characteristics. If more studies about genes that were differently expressed in each PDL cells & gingival fibroblasts would be performed in the future, it would be expected that the characteristics of PDL cells would be more clear.

• 한국발생생물학회지:발생과생식
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• 제23권3호
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• pp.285-295
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• 2019

Junction-mediating and regulatory protein (JMY) is a regulator of both transcription and actin filament assembly. The actin-regulatory activity of JMY is based on a cluster of three actin-binding Wiskott-Aldrich syndrome protein homology 2 (WH2) domains that nucleate actin filaments directly and promote nucleation of the Arp2/3 complex. In addition to these activities, we examined the activity of JMY generation in early embryo of mice carrying mutations in the JMY gene by CRISPR/Cas9 mediated genome engineering. We demonstrated that JMY protein shuttled expression between the cytoplasm and the nucleus. Knockout of exon 2, CA (central domain and Arp2/3-binding acidic domain) and NLS-2 (nuclear localization signal domain) on the JMY gene by CRISPR/Cas9 system was effective and markedly impeded embryonic development. Additionally, it impaired transcription and zygotic genome activation (ZGA)-related genes. These results suggest that JMY acts as a transcription factor, which is essential for the early embryonic development in mice.

• Molecules and Cells
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• 제38권9호
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• pp.789-795
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• 2015

A chromosome territory is composed of chromosomal subdomains. The internal structure of chromosomal subdomains provides a structural framework for many genomic activities such as replication and DNA repair, and thus is key to determining the basis of their mechanisms. However, the internal structure and regulating proteins of a chromosomal subdomain remains elusive. Previously, we showed that the chromosome territory expanded after BAF53 knockdown. Because the integrity of chromosomal subdomains is a deciding factor of the volume of a chromosome territory, we examined here the effect of BAF53 knockdown on chromosomal subdomains. We found that BAF53 knockdown led to the disintegration of histone H2B-GFP-visualized chromosomal subdomains and BrdU-labeled replication foci. In addition, the size of DNA loops measured by the maximum fluorescent halo technique increased and became irregular after BAF53 knockdown, indicating DNA loops were released from the residual nuclear structure. These data can be accounted for by the model that BAF53 is prerequisite for maintaining the structural integrity of chromosomal subdomains.

• Reproductive and Developmental Biology
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• 제37권4호
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• pp.169-174
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• 2013

Sperm capacitation refers to polymerization of filamentous (F)-actin from globular (G)-actin. While the role of actin-related protein 2/3 (Arp2/3) complex in actin polymerization is well appreciated, the underlying mechanism(s) and its relationship with capacitation are poorly understood. Therefore, to evaluate the potential role of Arp2/3 complex on capacitation, bovine spermatozoa were incubated with multiple doses (1, 10 and $100{\mu}M$) of CK-636, an inhibitor of Arp2/3 complex with heparin. The cellular localization of the Arp2/3 complex in spermatozoa was identified by immunohistochemistry, whereas western blot was also applied to detect the protein tyrosine phosphorylation of sperm proteins. Additionally, sperm motility and kinematic parameters were evaluated using a computer-assisted sperm analysis system. CK-636 resulted in significant changes in the ratio of Arp2/3 complex localization between acrosome and equatorial region of the spermatozoa. Short-term exposure of spermatozoa to $100{\mu}M$ of CK-636 significantly decreased sperm motility, however a non-detectable effect on protein tyrosine phosphorylation was observed during capacitation. On the basis of these results, we propose that Arp2/3 complex is associated with morphological changes during capacitation and compromised sperm motility.

• 대한물리치료과학회지
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• 제11권1호
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• pp.20-27
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• 2004

It is widely accepted that smooth muscle contraction is triggered by intracellular $Ca^{2+}$ ($[Ca^{2+}]_i$) released from intracellular $Ca^{2+}$ stores such as sarcoplasmic reticulum (SR) and from the extracellular space, The increased $[Ca^{2+}]_i$ can phosphorylate the 20-kDa myosin light chain ($MLC_{20}$) by activating MLC kinase (MLCK), and this initiates smooth muscle contraction. In addition to the $[Ca^{2+}]_i$-MLCK-tension pathway, a number of intracellular signal molecules, including mitogen-activated protein kinase (MAPK), protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K), and Rho-associated coiled coil-forming protein kinase (ROCK), play important roles in the regulation of smooth muscle contraction. However, the mechanisms regulating contraction of caldesmon (CaD), actin-binding protein, are not entirely elucidated in the presence of $Ca^{2+}$. It is known that CaD tightly interacts with actin and inhibits actomyosin ATPase activity. Therefore, the purpose of the present study was to investigate the roles of $Ca^{2+}$-dependent CaD in smooth muscle contraction. Endothelin-1 (ET-1), G-protein coupled receptor agonist and vasoconstrictor, increased both vascular smooth contraction and phosphorylation of CaD in the presence of $Ca^{2+}$. These results suggest that ET-1 induces contraction and phosphorylation of CaD in rat aortic smooth muscle, which may he mediated by the increase of $[Ca^{2+}]_i$.

• 한국자원식물학회지
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• 제28권5호
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• pp.648-655
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• 2015

본 연구에서는 도라지의 품종 구분 및 유전적 다양성 분석에 활용될 수 있는 분자표지 개발을 위하여 RAPD 마커를 활용하여 분석하였으며, 동시에 actin유전자 염기서열에서 유래한 분자표지를 제작하였다. 총 30개의 RAPD 분자표지를 활용한 분석을 진행하였으나, 재현성과 안정성이 확보된 DNA 상의 다형성은 확보할 수 없었다. 이에, 도라지 actin (Pg-actin) 유전자에 존재하는 Single Nucleotide Polymorphism (SNP)을 이용하여 품종 간 구분에 활용 가능한 분자마커로의 전환을 탐색하였다. 도라지 genomic DNA에 actin 유래 유전자를 사용하여 2개의 actin homologs를 확보하였으며, 이를 염기서열 분석하여 3.4 kb의 Pg-actin fragment와 Pg-actin과 28.6%의 유사도를 가진 1.4 kb의 actin homologue를 획득하였다. 획득된 Pg-actin은 4개의 exon과 3개의 intron으로 구성된 유전자로, DNA 다형성 탐색을 통해 intron 3의 286 bp 위치에서 SNP (G ↔ A)를 발견하였으며, 이를 활용도 높은 CAPS marker로 전환하여 PgActin-Int3 마커를 개발하였다. Pg-Actin-Int3 마커를 32개의 도라지 유전자원에 적용시켜 본 결과 품종 간의 차이를 보이는 부분이 확인되었다. 본 연구에서 확보된 도라지 DNA 염기서열정보는 도라지의 유전적 다양성 분석 및 도라지 분자육종에 활용될 수 있을 것이라 전망된다.

• The Korean Journal of Physiology and Pharmacology
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• 제14권4호
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• pp.205-212
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• 2010

Endometritis is one of the primary reasons for reproductive failure. In order to investigate endometritis-associated marker proteins, proteomic analysis was performed on bovine endometrium with endometritis. In bovine endometritis, desmin, $\alpha$-actin-2, heat-shock protein (HSP) 27, peroxiredoxin-6, luteinizing hormone receptor isoform 1, collectin-43 precursor, deoxyribonuclease-I (DNase-I), and MHC class I heavy chain (MHC-Ih) were up-regulated. In contrast, transferrin, interleukin-2 precursor, hemoglobin $\beta$ subunit, and potassium channel tetramerisation domaincontaining 11 (KCTD11) were down-regulated in comparison to normal endometrium. The proteomic results were validated by semiquantitative-PCR and immunoblot analysis. The mRNA levels of desmin, transferrin, $\alpha$-actin-2, HSP27, KCTD11, and MHC-Ih were up-regulated by over 1.5-fold, and showed a pattern similar to their proteomic profiles. Desmin and $\alpha$-actin-2 protein showed positive correlations between proteomic analysis and immunoblot analysis. These results suggest that desmin and $\alpha$-actin-2 may play important roles in endometritis-related function, and could be useful markers for the diagnosis of bovine endometritis.

• BMB Reports
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• 제43권12호
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• pp.795-800
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• 2010

Huntingtin-interacting protein 1-related (HIP1r) is known to function in clathrin-mediated endocytosis and regulation of the actin cytoskeleton, which occurs continuously in non-dividing cells. This study reports a new function for HIP1r in mitosis. Green fluorescent protein-fused HIP1r localizes to the mitotic spindles. Depletion of HIP1r by RNA interference induces misalignment of chromosomes and prolonged mitosis, which is associated with decreased proliferation of HIP1r-deficeint cells. Chromosome misalignment leads to missegregation and ultimately production of multinucleated cells. Depletion of HIP1r causes persistent activation of the spindle checkpoint in misaligned chromosomes. These findings suggest that HIP1r plays an important role in regulating the attachment of spindle microtubules to chromosomes during mitosis, an event that is required for accurate congression and segregation of chromosomes. This finding may provide new insights that improve the understanding of various human diseases involving HIP1r as well as its fusion genes.

• 한국임상수의학회지
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• 제39권2호
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• pp.51-58
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• 2022

Quercetin, a flavonoid found in fruits and vegetables, exhibits a strong anti-inflammatory activity. The objective of this study was to examine the effect of quercetin on chemotactic activity of peripheral blood polymorphonuclear cells (PMNs) to culture supernatant from peripheral blood mononuclear cells (PBMCs) stimulated with lipopolysaccharide (LPS). In addition, we determined whether this effect is related to interleukin (IL)-8 and changes in cytoskeleton. The chemotactic activity of PMNs was evaluated by a modified Boyden chamber assay. Total cellular filamentous (F)-actin levels were measured by method of fluorescence microscopy. The levels of IL-8 mRNA and protein were measured by real time polymerase reaction method and enzyme-linked immunosorbent assay, respectively. Quercetin (0-50 µM) itself has no chemoattractant effect for PMNs. The culture supernatant from PBMCs (2 × 10⁶ cells/mL) treated with LPS (1 ㎍/mL) showed remarkable increase in chemotaxis of PMNs. However, this effect was reduced dose-dependently by treatment with quercetin. In addition, PBMCs treated with LPS revealed enhanced levels in IL-8 protein and mRNA. Co-treatment of LPS with quercetin (50 µM) in PBMCs decreased IL-8 production and expression. Treatment of quercetin (0-50 µM) on PMNs to rpIL-8 (10 nM) decreased dose-dependently the chemotactic activity of PMNs. Treatment of quercetin on PMNs to IL-8 also reduced their total cellular F-actin level. These results suggested that quercetin attenuates chemotactic activity of PMNs, which is mediated by down-regulation of IL-8 production from LPS-stimulated PBMCs and inhibition of F-actin polymerization in PMNs.