Tissue Inhibitor of Metalloproteinase-1 Pro-motes NIH3T3 Fibroblast Proliferation by Activating p-Akt and Cell Cycle Progression

Yang Lu,Shuxin Liu,Shujia Zhang,Guangyan Cai,Hongwei Jiang,Huabin Su,Xiaofan Li,Quan Hong,Xueguang Zhang,Xiangmei Chen 한국분자세포생물학회 2011 Molecules and cells Vol.31 No.3

Tissue inhibitor of metalloproteinase-1 (TIMP-1) plays various roles in cell growth in different cell types. However, few studies have focused on TIMP-1’s effect on fibroblast cells. In this study, we investigated the effects of TIMP-1 overexpression on NIH3T3 fibroblast proliferation and potential transduction signaling pathways involved. Overexpression of TIMP-1, by transfection of the pLenti6/ V5-DESTTIMP-1 plasmid, significantly promoted NIH3T3 proliferation as determined by the BrdU array. Neither 5 nor 15 nM GM6001 (matrix metalloproteinase system inhibitor) affected NIH3T3 proliferation, but 45 nM GM6001 inhibited proliferation. TIMP-1 overexpression activated the p-Akt pathway, but not the p-ERK or p-p38 pathway. In TIMP-1-transfected cells, cyclinD1 was upregulated and p21CIP1 and p27^(KIP1) were downregulated, which promoted cell entry into the S and G2/M phases. The PI3-K inhibitor LY294002 abolished the TIMP-1-induced effects. Overexpression of intracellular TIMP-1 stimulated NIH3T3 fibroblast proliferation in a matrix metalloproteinase (MMP)-independent manner by activating the p-Akt pathway and related cell cycle progression.

NIH/3T3 Cell을 사용한 임피던스 바이오센서의 패턴 너비와 간격에 따른 주파수 의존도

김예은,최지수,강다현,김용준,나지균,권정민,정지용,정재훈,이가영,장문규 한국물리학회 2022 새물리 Vol.72 No.9

This research presents impedance biosensors that can monitor the growth and drug reaction of NIH/3T3 cells in real time through semiconductor processes. Biosensors of 163, 211, 300, and 518 μm were fabricated in four different width/spacings. The change in capacitance with impedance was observed. According to the width and spacing of each sensor, the most sensitive frequency band was determined by measuring the capacitance-frequency through a frequency sweep at 1 k–1 MHz. Results showed that the 163, 211, 300, and 518 μm sensors had sensitive frequency at the range of 411–489, 367–440 and 221–274 kHz, respectively. After that, 1–800 kHz capacitance-time data was measured. The capacitance values were compared with the sensitive frequency range and outside range. The finding showed that the larger the width and spacing of the patterns, the more sensitive the sensors are in the low frequency range. The most accurate capacitance values were measured in the sensitive frequency range. This study showed the importance of frequency selection and the width/spacing design of electrodes relative to the frequency range to improve sensitivity in electrical impedance measurement. 본 연구는 NIH/3T3 세포의 성장 및 사멸과정을 실시간으로 모니터링 할 수 있는 임피던스 바이오센서를 반도체공정을 통하여 제작하였다. 163, 211, 300, 518 μm 로 4가지의 다른 너비/간격으로 제작하였다. 측정되는 임피던스 값에서 정전용량의 변화량을 관찰하였고, 1 k–1 MHz에서 주파수 휩씀을 통해 정전용량-주파수 데이터를 측정하여 각 센서의 너비/간격에 따른 가장 민감한 주파수 영역대를 알 수 있었다. 그 결과 163과 211 μm 센서는 411–489 kHz, 300 μm는 367–440 kHz, 518 μm는 221–274 kHz에서 민감한 주파수 대역을 확인하였다. 이후 1–800 kHz 정전용량-시간 데이터를 측정하였다. 민감한 주파수 범위와 그 범위 외의 정전용량 변화량을 비교하였다. 그 결과 패턴의 너비/간격이 증가할수록 낮은 주파수 영역대에서 민감하다는 것을 확인하였고, 민감한 주파수 영역대에서 측정된 정전용량의 변화량이 가장 정확하게 반영된 것을 관찰하였다. 본 연구는 전기 임피던스 측정 시 민감도 향상을 위하여 측정하는 주파수 선정의 중요성과 측정하는 주파수 영역에 따라 전극의 너비/간격 설계의 필요성을 제시한다.

Transient activation of AMP-activated protein kinase at G1/S phase transition is required for control of S phase in NIH3T3 cells

Park, In-Ja,Tran, Quynh Hoa,Amin, Ain Syafiza Mohd,Chu, Thanh Lan,Yang, Goowon,Choe, Wonchae,Kang, Insug,Kim, Sung Soo,Ha, Joohun Elsevier 2018 Biochemical and biophysical research communication Vol.504 No.2

<P><B>Abstract</B></P> <P>AMP-activated protein kinase (AMPK) functions as a cellular energy sensor by monitoring the cellular AMP:ATP ratio and plays a central role in cellular and whole-body energy homeostasis. Recent studies have suggested that AMPK also contribute to cell cycle regulation, but its role in this field remains almost elusive. In the present study, we report that AMPKα1 was transiently activated during G<SUB>1</SUB>/S transition phase in NIH3T3 cells in the absence of any metabolic stress. Inhibition of AMPK activity at G<SUB>1</SUB>/S transition phase completely blocked cells from entering S phase; in contrast, persistent activation of AMPK at G<SUB>1</SUB>/S transition phase allowed cells to normally enter S phase, but these cells failed to proceed to G<SUB>2</SUB>/M phase, stacking at S phase. We further demonstrated that activation of AMPK at G<SUB>1</SUB>/S transition phase depends on Ca<SUP>2+</SUP> transients and CaMKKβ activity, but not on energy status. Collectively, these data indicate that temporal regulation of AMPK is required for proper control of S phase in NIH3T3 cells.</P> <P><B>Highlights</B></P> <P> <UL> <LI> AMPK is transiently activated at G1/S phase of NIH3T3 cell cycle. </LI> <LI> Temporal activation of AMPK is required for control of S phase. </LI> <LI> AMPK activation at G1/S phase depends on intracellular calcium and CaMKKbeta. </LI> </UL> </P>

Ag-NORs 염색법과 [³H]thymidine incorporation법에 의한 caffeine의 NIH3T3 세포증식 억제효과 비교측정

김성호,이차수,Kim, Sung-ho,Lee, Cha-soo 대한수의학회 1990 大韓獸醫學會誌 Vol.30 No.4

Inhibitory effect of caffeine on NIH3T3 cell proliferation was studied by using [$^3H$]thymidine incorporation and a modified one-step silver staining technique. The latter technique shows argyrophilic nucleolar organizer region-associated proteins (Ag-NORs), which are seen in nuclei as black dots. The result was compared with the counts of [$^3H$] thymidine incorporation. The results were summarized as follows; 1. The Ag-NORs numbers of NIH3T3 cells were $6.81{\pm}1.38$ at 24 hrs, $7.13{\pm}1.26$ at 48 hrs, $8.50{\pm}2.04$ at 72 hrs after incubation. 2. The numbers of Ag-NORs were significantly decrease in caffeine treated groups (p<0.01). 3. The counts of [$^3H$] thymidine incorporation were similar to the result of using Ag-NORs staining technique. It is concluded that Ag-NOR is a rapid, simple and compatible method to quantitate cell proliferation as compared with [$^3H$]thymidine incorporation.

Over-Expression of Phospholipase D Isozymes Down-Regulates Protein Kinase CKII Activity via Proteasome-Dependent CKII Degradation in NIH3T3 Cells

윤수현,민도식,Young-Seuk Bae 한국분자세포생물학회 2009 Molecules and cells Vol.27 No.3

Over-expression of phospholipase D (PLD) 1 or PLD2 downregulated CKII activity in NIH3T3 cells. The same results were found with catalytically inactive mutants of PLD isozymes, indicating that the catalytic activity of PLD is not required for PLD-mediated CKII inhibition. Consistent with this, 1-butanol did not alter CKII activity. The reduction in CKII activity in PLD-over-expressing NIH3T3 cells was due to reduced protein level, but not mRNA level, of the CKIIβ subunit. This PLD-induced CKIIβ degradation was mediated by ubiquitin-proteasome machinery, but MAP kinase and mTOR were not involved in CKIIβ degradation. PLD isozymes interacted with the CKIIβ subunit. Immunocytochemical staining revealed that PLD and CKIIβ colocalize in the cytoplasm of NIH3T3 cells, especially in the perinuclear region. PLD binding to CKIIβ inhibited CKIIβ autophosphorylation, which is known to be important for CKIIβ stability. In summary, the current data indicate that PLD isozymes can down-regulate CKII activity through the acceleration of CKIIβ degradation by ubiquitin-proteasome machinery.

갈색거저리 유충 추출물의 항산화 활성 및 모발 성장 촉진 효과

백민희(Minhee Baek),서민철(Minchul Seo),김미애(Mi-Ae Kim),윤은영(Eun-Young Yun),황재삼(Jae-Sam Hwang) 한국생명과학회 2017 생명과학회지 Vol.27 No.11

최근 들어 곤충을 식품 및 바이오 소재로 이용한 연구가 활발히 진행되고 있다. 그러나 곤충을 이용한 모발성장 효과에 대한 연구는 아직 미흡한 실정이다. 따라서 본 연구에서는 탈모 예방 및 모발 성장 효과를 가진 새로운 천연물 소재 개발을 위해 갈색거저리 유충 추출물의 항산화 활성 및 모발 성장 촉진 효과를 연구하였다. 갈색거저리 유충 추출물의 항산화 활성 평가를 위해서 DPPH 라디칼 및 아질산염 소거능을 측정하였다. 모발 성장촉진 효과를 측정하기 위해서는 인간 모유두세포(human dermal papilla cell)와 섬유아세포(fibroblast, NIH3T3 cell)를 이용하였으며 MTS assay를 통해 세포생존율 및 세포증식률을 측정하였다. 모유두세포에서 dihydrotesteone (DHT)에 의한 세포사 억제 효과를 확인하였으며, 섬유아세포에서는 tolbutamide (TBM)의 potassium channel blocker 역할에 의한 세포사 억제 효과를 확인하였다. DPPH radical 및 아질산염 소거능 측정 결과 갈색거저리 유충 추출물은 항산화 역할이 뛰어난 것으로 보고된 블루베리와 유사하거나 높은 정도의 항산화능을 가지는 것으로 확인되었다. In vitro 상에서 갈색거저리 유충 추출물을 48시간 동안 처리한 경우, 모유두세포와 섬유아세포의 세포증식을 218% 및 116%까지 증가시켰다. 또한, 모유두세포에서 DHT 처리에 의한 세포사가 갈색거저리 유충추출물에 의해 억제되는 것을 확인하였으며, 섬유아세포에서는 potassium channel blocker인 TBM에 의해 세포생존율이 감소하였으나 갈색거저리 유충 추출물 처리 시 세포생존율이 정상군과 비슷한 정도로 회복되는 것을 확인하였다. 이상의 결과로부터 갈색거저리 유충 추출물을 이용한 모발성장 및 탈모방지 기능성 소재 개발 가능성을 확인하였다. Tenebrio molitor samples were investigated as novel biomaterials and sources of food in several recent studies. However, the insects" effects on hair growth were not sufficiently researched. To develop novel and natural materials for preventing alopecia and promoting hair growth, this study investigated the antioxidant activities and hair-growth promotion effects of TMEs. To determine the antioxidant activities, the TMEs" DPPH radical- and nitrite-scavenging activities were examined. To determine hair-growth promotion effects, proliferations of human dermal papilla cells (DPCs) and the murine fibroblast cell line NIH3T3 were evaluated by using an MTS assay. In addition, estimations were made for cell viabilities against cell death induced by dihydrotesterone (DHT) in DPCs and inhibitory effects against potassium channel blocking induced by tolbutamide (TBM) in NIH3T3 cells. The DPPH radical scavenging activity was 81.17%, and the nitrite scavenging activity was 43.69%; the activities were similar to the activities of blueberry extracts. Moreover, the TMEs promoted the proliferation of human DPCs and NIH3T3 cells, which were concentrated dependently. The TMEs prevented not only DHT-induced DPC cytotoxicity but also TBM"s action as a potassium channel blocker in NIH3T3 cells. The results suggested that TME could be used as a functional therapeutic alopecia reagent, to prevent hair loss and to promote hair growth.

Induction of MAP kinase phosphatase 3 through Erk/MAP kinase activation in three oncogenic Ras (H-, K- and N-Ras)-expressing NIH/3T3 mouse embryonic fibroblast cell lines

( Jaehyung Koo ),( Sen Wang ),( Nana Kang ),( Sun Jin Hur ),( Young Yil Bahk ) 생화학분자생물학회 2016 BMB Reports Vol.49 No.7

Ras oncoproteins are small molecular weight GTPases known for their involvement in oncogenesis, which operate in a complex signaling network with multiple effectors. Approximately 25% of human tumors possess mutations in a member of this family. The Raf1/MEK/Erk1/2 pathway is one of the most intensively studied signaling mechanisms. Different levels of regulation account for the inactivation of MAP kinases by MAPK phosphatases in a cell type- and stimuli-dependent manner. In the present study, using three inducible Ras-expressing NIH/3T3 cell lines, we demonstrated that MKP3 upregulation requires the activation of the Erk1/2 pathway, which correlates with the shutdown of this pathway. We also demonstrated, by applying pharmacological inhibitors and effector mutants of Ras, that induction of MKP3 at the protein level is positively regulated by the oncogenic Ras/Raf/MEK/Erk1/2 signaling pathway. [BMB Reports 2016; 49(7): 370-375]

NF-κB signaling is key in the wound healing processes of silk fibroin

Park, Ye Ri,Sultan, Md. Tipu,Park, Hyun Jung,Lee, Jung Min,Ju, Hyung Woo,Lee, Ok Joo,Lee, Dong Jin,Kaplan, David L.,Park, Chan Hum Elsevier Science B.V. Amsterdam 2018 ACTA BIOMATERIALIA Vol. No.

<P><B>Abstract</B></P> <P>Silk fibroin (SF) is a well-studied biomaterial for tissue engineering applications including wound healing. However, the signaling mechanisms underlying the impact of SF on this phenomenon have not been determined. In this study, through microarray analysis, regulatory genes of NF-ĸB signaling were activated in SF-treated NIH3T3 cells along with other genes. Immunoblot analysis confirmed the activation of the NF-ĸB signaling pathway as SF induced protein expression levels of IKKα, IKKβ, p65, and the degradation of IκBα. The treatment of NIH3T3 cells with SF also increased the expression of cyclin D1, vimentin, fibronectin, and vascular endothelial growth factor (VEGF). The expression of these factors by SF treatment was abrogated when NF-ĸB was inhibited by a pharmacological inhibitor Bay 11-7082. Knockdown of NF-ĸB using siRNA of IKKα and IKKβ also inhibited the SF-induced wound healing response of the NIH3T3 cells in a wound scratch assay. Collectively, these results indicated that SF-induced wound healing through the canonical NF-κB signaling pathway via regulation of the expression of cyclin D1, vimentin, fibronectin, and VEGF by NIH3T3 cells. Using an <I>in vivo</I> study with a partial-thickness excision wound in rats we demonstrated that SF-induced wound healing via NF-κB regulated proteins including cyclin D1, fibronectin, and VEGF. The <I>in vitro</I> and <I>in vivo</I> data suggested that SF induced wound healing via modulation of NF-ĸB signaling regulated proteins.</P> <P><B>Statement of Significance</B></P> <P>Silk fibroin has been effectively used as a dressing for wound treatment for more than a century. However, mechanistic insight into the basis for wound healing via silk fibroin has not been elucidated. Here we report a key mechanism involved in silk fibroin induced wound healing both <I>in vitro</I> and <I>in vivo.</I> Using genetic- and protein-level analyses, NF-κB signaling was found to regulate silk fibroin-induced wound healing by modulating target proteins. Thus, the NF-κB signaling pathway may be utilized as a therapeutic target during the formulation of silk fibroin-based biomaterials for wound healing and tissue engineering.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

천연추출물의 발모촉진 및 탈모 예방에 미치는 영향

배상옥 ( Sang-ok Bae ),오강수 ( Gang-su Oh ) 한국미용예술경영학회 2022 미용예술경영연구 Vol.16 No.2

본 연구는 검은콩, 은행, 녹차를 포함한 천연추출물이 두발의 발모 촉진과 두개피부에 미치는 영향을 조사한 연구이다. 연구 방법은 검은콩, 건조은행, 녹차잎을 2 : 1 : 1의 비율로 10 배수의 증류수에 침지하고 100℃에서 2시간 열수추출한 후, Eclipse Plus C18(4.6×250 mm, 5 μm)를 사용하여, 이동상은 초순수로 희석한 70% Methanol을 이용하였다. 추출물의 항균 활성은 Malassezia Furfur를 이용하였으며 항산화 활성은 DPPH에 대한 전자공여능 측정으로 평가하였다. 인간 모유두세포 및 섬유아세포의 증식 활성은 96 Well Plate에 세포를 3×104 (100 ㎕/well)로 분주하고 24시간 배양한 후 MTT Assay를 실시하였다. 천연추출물을 농도별로 투여하고 배양이 끝나면 DMSO 용액을 넣고, Plate Shaker에서 30분 추출 후, 570 nm에서 ELISA를 이용하여 흡광도를 측정하였다. 연구 결과 항균 효과 조사에서 천연추출물의 모낭염 유발균인 Pityrosporum Ovale와 지루성피부염 유발균인 Malassezia Furfur에 대해 성장억제를 확인하였고, DPPH 라디컬 소거능 측정을 통해 항산화 활성을 확인하였다. 또한, 인간 모유두세포(HFDPC)와 섬유아세포(NIH3T3)의 세포증식 효과에 대한 실험에서 천연추출물 0.10% 농도에서 모유두세포의 40% 이상 증식 효과, 섬유아세포는 0.02% 농도에서 약 10% 증식 효과가 있음을 확인하였다. 이들 3종의 천연추출물은 발모 촉진 및 탈모 예방 효과를 목적으로 하는 두개피부 및 모발보호 제품개발을 위한 기초자료로 활용될 수 있음을 시사하는 바이다. This study examined the effects of natural extracts including black bean, Ginkgo biloba L. fruits, and green tea, on hair growth and scalp improvement. For the research method, black beans, dried ginkgo biloba, and green tea leaves were immersed in 10 times distilled water at a ratio of 2 : 1 : 1, extracted with hot water at 100 ° C for 2 hours, and Eclipse Plus C18 (4.6 × 250 mm, 5 μm) was used. As the mobile phase, 70% methanol diluted with ultrapure water was used. The antibacterial activity of the extract was evaluated using Malassezia furfur, and the antioxidant activity was evaluated by measuring the electron donating ability to DPPH. For proliferative activity of human dermal papilla cells and fibroblasts, 3×104 (100 μl/well) of cells were aliquoted in a 96 well plate and cultured for 24 hours, followed by MTT assay. After administration of natural extracts by concentration, and after incubation, DMSO solution was added, and after 30 minutes extraction on a plate shaker, absorbance was measured using ELISA at 570 nm. Antimicrobial activity of natural extracts on Pityrosporum ovale and Malassezia furfur were investigated. In antioxidative activities of natural extracts, DPPH radical scavenging activity was about 62.40±4.8 % at 5.0 mg/mL. On the other hand, cell proliferation in NIH3T3 cells and HFDPC cells were determined. Natural extracts promoted the proliferation of NIH3T3 cells, a human epidermal cells by about 10 % at 0.02 % concentration. HFDPC cells, a hair follicle dermal papilla cells were significantly stimulated in cell proliferation by above 40% at 0.10% concentration. These results suggest that natural extracts have antimicrobial activity, antioxidant effects, induce cell proliferation, and could be used for hair loss prevention and scalp improvement.

다중 웰 어레이 바이오센서를 위한 패턴 크기 별 정전용량 변화 분석: ECIS 시스템을 활용한 세포 거동 연구

강다현,김석규,남수권,장문규 한국물리학회 2023 새물리 Vol.73 No.10

ECIS 시스템을 기반으로 하는 바이오센서를 제작하여 NIH/3T3 세포의 성장과정과 퓨로마이신 약물반응에 대한 정전용량을 측정하였다. 이때 센서의 패턴 사이즈의 변화가 정전용량 측정에 미치는 영향을 분석하기 위해 기존 패턴을 기준으로 확대 및 축소하여 바이오센서를 제작하고 측정을 수행하였다. 그 결과 100 kHz 이후에서 측정된 정전용량 변화 데이터가 세포의 구동을 올바르게 반영하였으며, 이는 패턴 사이즈에 관계없이 나타났다. 따라서 패턴사이즈의 변화가 정전용량 측정에 문제가 없음을 확인하고 축소된 패턴을 기준으로 다중 웰 어레이 바이오센서를 제작하였다. 동일한 측정을 수행한 결과 정전용량 변화 개형의 재현성과 웰 간의 데이터 균일성을 확보하였으며, 이를 통해 웰 사이 조건을 변경하여 독립적인 데이터를 동시에 얻는 실험을 진행하거나 3 × 3 등으로 배열하는 패턴의 수를 늘리는 등 다중 웰 바이오센서의 발전 가능성을 확인하였다. A biosensor was fabricated based on the ECIS system to measure the capacitance of NIH/3T3 cells during cell growth and drug reactions. At this time, the biosensors were fabricated by enlarging and reducing the existing pattern to analyze the effect of changing the sensor pattern size on capacitance measurement. Following the capacitance measurement, the capacitance change data, measured at a frequency larger than 100 kHz, correctly reflected the cell state, which occurred regardless of the pattern size. Therefore, the results confirmed that no problems were encountered in measuring capacitance due to pattern size variation, and 3 × 3 array biosensors were fabricated based on the reduced pattern. The same measurement ensured the reproducibility of the capacitance values and uniformity of data between wells.