• Toxicological Research
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• 제22권3호
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• pp.283-291
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• 2006

The survival and growth of epithelial cells depends on adhesion to the extracellular matrix. An adhesion signal may regulate the initiation of differentiation, since epidermal keratinocytes differentiate as they leave the basement membrane. A metabolically dead cornified cell envelope is the end point of epidermal differentiation so that this process may be viewed as a specialized form of programmed cell death. In order to investigate the precise cellular signaling events loading to terminal differentiation of keratinocytes, we have utilized HaCaT cells to monitor the biological consequences of $Ca^{2+}$ stimulation and numerous downstream signaling pathways, including activation of the extracellular signal-regulated protein kinase(ERK) pathway and activation of phosphatidylinositol 3-kinase(PI3K). The results presented in this study show that $Ca^{2+}$ function as potent agents for the differentiation of HaCaT keratinocytes, and this differentiation depends or the activation of ERK, Protein kinase B(PKB) and p70 ribosomal protein S6 kinase(p70S6K). Finally, the results show that the expression of Activator protein 1(AP-1; c-Jun and c-Fos) increased following $Ca^{2+}$-mediated differentiation of HaCaT cells, suggesting that ERK-mediated AP-1 expression is critical for initiating the terminal differentiation of keratinocytes.

• Asian Pacific Journal of Cancer Prevention
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• 제13권6호
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• pp.2527-2532
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• 2012

Objectives: The aim of the present study was to explore mechanisms underlying the effects of down-regulating ${\beta}$-catenin expression on esophageal carcinoma (EC) cells. Methods: Cell cycle distribution and apoptosis were determined using flow cytometry and annexin V apoptosis assay, respectively. Transmission electron microscopy (TEM) was used to examine changes in ultrastructure, while expression of cyclin D1 protein and mRNA was detected by western blot and real-time PCR. Proliferating cell nuclear antigen (PCNA) and extracellular signal-regulated kinase (ERK) 1-2 were evaluated by Western blot analysis. PCNA labeling index (LI) was determined by immunocytochemistry. Results: Compared with pGen-3-con transfected and Eca-109 cells, the percentage of G0/G1-phase pGen-3-CTNNB1 transfected cells was obviously increased (P<0.05), with no significant difference among the three groups with regard to apoptosis (P>0.05). pGen-3-CTNNB1 transfected cells exhibited obvious decrease in cyclin D1 mRNA and protein expression (P<0.05) and the ultrastructure of Eca-109 cells underwent a significant change after being transfected with pGen-3-CTNNB1, suggesting that down-regulating ${\beta}$-catenin expression can promote the differentiation and maturation. The expression of PCNA and the ERKI/2 phosphorylation state were also down-regulated in pGen-3-CTNNB1 transfected cells (P<0.05). At the same time, the PCNA labeling index was decreased accordingly (P<0.05). Conclusion: Inhibition of EC Eca-109 cellproliferation by down-regulating ${\beta}$-catenin expression could improve cell ultrastructure by mediating blockade in G0/G1 through inhibiting cyclin D1, PCNA and the MAPK pathway (p-ERK1/2).

• 한국응용곤충학회지
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• 제45권1호
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• pp.15-24
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• 2006

프루텔고치벌(Cotesia plutellae)은 내부기생봉이고 kB억제자 (IkB)와 유사한 유전자가 이 기생봉의 절대 공생바이러스(C. plutellae bracovirus: CpBV) 게놈에서 발견되었다. 이 유전자의 발현 부위는 417 br의 크기이며 138개 아미노산 서열 정보를 포함하였다. 이 단백질은 4개의 ankyrin 반복영역을 지니고 있었으며, 알려진 다른 폴리드나바이러스 유래 IkB 유전자와 높은 상동성을 보였다. 초파리 Cactus 단백질을 통해 대상 기주 IkB와 비교하여 보면, IkB 신호수신영역이 부재하는 구조를 보여, CpBV-IkB는 NFkB 신호전달체계의 비가역적 억제 인자로 작용할 것으로 추정되었다. CpBV-IkB는 프루텔고치 벌에 기생된 배추좀나방에서만 발현되었다. 정량적 RT-PCR 방법으로 CpBV-IkB의 발현량을 조사하여 보면, 기생 첫날부터 발현을 보이기 시작하여 뚜렷한 발현량을 기생 전체 기간동안 유지하는 양상을 보였다. 이 CpBV-IkB의 기능 분석이 간접적으로 이뤄졌으며, 이 유전자 발현물이 대상 기주 항바이러스 억제 인자로 작용할 것이라는 가설을 제시하였다.

• 생명과학회지
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• 제28권1호
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• pp.43-49
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• 2018

Chrysoeriol은 alfalfa에서 주로 발견되는, 식물계에 많이 분포하고 있는 flavone으로 전통의학에서 소화불량, 천식, 비뇨기계 이상의 치료에 사용되어 왔다. 최근의 연구에서는 항염증 효과가 있는 것으로 밝혀졌으나 항산화 효과에 대한 분석은 없었다. 본 연구에서는 chrysoeriol의 항산화 효과와 그 분자적 기전을 RAW 264.7 cell에서 세포생존율, reactive oxygen species (ROS)와 Western blot분석을 통해 알아보고자 하였다. Chrysoeriol은 lipopolysaccharide(LPS)에 의해 발생한 ROS를 세포독성없이 농도의존적으로 제거하였다. 그리고 항산화효과를 보이는 2상 효소 중 하나인 heme oxygenase (HO)-1의 발현을 강하게 유도하였고, 그와 동시에 전사인자인 Nrf2의 핵내 이동도 촉진하는 것으로 밝혀졌다. 특히, 산화스트레스에 대한 세포내 산화환원항상성 유지에 중요한 역할을 하고 있는 것으로 알려진 mitogen activated protein kinase (MAPK)와 phosphoinositide 3-kinase (PI3K)의 분석결과, chrysoeriol은 extracellular signal regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK)와 p38의 인산화를 통해 HO-1의 발현을 유도하는 것으로 나타났다. HO-1에 의한 항산화 효과를 확인하기 위하여 chrysoeriol을 전처리한 후 t-BHP에 의한 산화 스트레스에 세포를 노출시킨 결과, chrysoeriol 처리에 의해 세포사멸이 줄어드는 것을 확인하였고, HO-1의 유도제와 억제제의 처리에 따라 세포생존율 또한 조절되는 것을 확인할 수 있었다. 따라서, chrysoeriol은 HO-1의 발현을 유도하여 항산화 효과를 높이고 이것은 Nrf2/MAPK 신호전달 체계에 의한다는 것을 알 수 있었다.

• Molecules and Cells
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• 제37권12호
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• pp.899-906
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• 2014

Prostaglandin $E_2$ ($PGE_2$) promotes tumor-persistent inflammation, frequently resulting in cancer. Curcumin is a diphenolic turmeric that inhibits carcinogenesis and induces apoptosis. $PGE_2$ inhibits curcumin-induced apoptosis; however, the underlying inhibitory mechanisms in colon cancer cells remain unknown. The aim of the present study is to investigate the survival role of $PGE_2$ and whether addition of exogenous $PGE_2$ affects curcumininduced cell death. HCT-15 cells were treated with curcumin and $PGE_2$, and protein expression levels were investigated via Western blot. Reactive oxygen species (ROS) generation, lipid peroxidation, and intracellular glutathione (GSH) levels were confirmed using specific dyes. The nuclear factor-kappa B ($NF-{\kappa}B$) DNA-binding was measured by electrophoretic mobility shift assay (EMSA). $PGE_2$ inhibited curcumin-induced apoptosis by suppressing oxidative stress and degradation of PARP and lamin B. However, exposure of cells to the EP2 receptor antagonist, AH6809, and the PKA inhibitor, H89, before treatment with $PGE_2$ or curcumin abolished the protective effect of $PGE_2$ and enhanced curcumin-induced cell death. $PGE_2$ activates PKA, which is required for cAMP-mediated transcriptional activation of CREB. $PGE_2$ also activated the Ras/Raf/Erk pathway, and pretreatment with PD98059 abolished the protective effect of $PGE_2$. Furthermore, curcumin treatment greatly reduced phosphorylation of CREB, followed by a concomitant reduction of $NF-{\kappa}B$ (p50 and p65) subunit activation. $PGE_2$ markedly activated nuclear translocation of $NF-{\kappa}B$. EMSA confirmed the DNA-binding activities of $NF-{\kappa}B$ subunits. These results suggest that inhibition of curcumin-induced apoptosis by $PGE_2$ through activation of PKA, Ras, and $NF-{\kappa}B$ signaling pathways may provide a molecular basis for the reversal of curcumin-induced colon carcinoma cell death.

• 생명과학회지
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• 제22권1호
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• pp.87-91
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• 2012

암을 유발하는 phorbol ester로 알려진 Phorbol 12-myristate 13-acetate (PMA)는 동물세포에서 신호전달 효소의 하나인 protein kinase C (PKC)를 활성화시킨다. 본 연구에서는 옥수수 일차뿌리에서 PMA가 에틸렌 생성을 통하여 굴중성 반응을 조절하는 효과를 연구하였다. PMA는 8시간 동안 $10^{-6}$ M과 $10^{-4}$ M에서 농도 의존적으로 뿌리 생장과 굴중성 반응을 촉진시켰다. 이러한 촉진 효과는 PKC의 억제제인 staurosporine (STA)에 의해 상쇄되었다. 이 결과는 굴중성 반응이 신호전달 체계에 관여하는 protein kinase C를 통하여 조절될 가능성을 제시하고 있다. 식물호르몬인 에틸렌도 뿌리 생장과 굴중성 반응에 중요한 역할을 한다고 알려져 있다. 에틸렌 생성은 $10^{-6}$ M과 $10^{-4}$ M PMA에 의하여 각각 26%와 37% 증가하였다. PMA는 또한 ACC synthase (ACS) 활성을 촉진시켰다. 또한 이 증가 효과는 STA에 의하여 상쇄되었다. 이 결과는 옥수수 뿌리에서 굴중성 반응은 에틸렌 생성을 거쳐 protein kinase를 통하여 조절될 가능성을 제시하고 있다.

• Asian Pacific Journal of Cancer Prevention
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• 제16권8호
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• pp.3117-3120
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• 2015

Objective: Interferon-${\gamma}$ (IFN-${\gamma}$) and signal transducers and activators of transcription (STATs) each play an important role in carcinogenesis associated with viral infection. Cervical cancer is almost invariably associated with infection by human papillomavirus (HPV), and previous studies suggested that dysregulation of the signal pathway involved in IFN-${\gamma}$ and STATs is associated. Our objective was to evaluate the association of SNPs in STAT2, STAT3, and IFN-${\gamma}$ with cervical cancer susceptibility in Chinese Han women in Hunan province. Materials and Methods: Genomic DNA was extracted from peripheral blood samples of 234 cervical cancer patients and 216 healthy female controls. STAT2 and STAT3 genotyping was performed using polymerase chain reaction-restriction enzyme (PCR-RE) analysis. IFN-${\gamma}$ genotyping was detected by PCR-amplification of specific allele (PASA). Results: For STAT2 rs2066807 polymorphisms, there was no significant difference of genotype distribution (P=0.827) and allele frequencies (P=0.830, OR=1.09, 95% CI: 0.51-2.31) between cases and controls. For STAT3 rs957970 polymorphisms, there was no significant difference of genotype distribution (P=0.455) and allele frequencies (P=0.560, OR=0.92, 95% CI: 0.71-1.20) between cases and controls. For IFN-${\gamma}$ +874A/T polymorphisms, there was no significant difference of genotype distribution (P=0.652) and allele frequencies (P=0.527, OR=1.12, 95% CI: 0.79-1.59) between cases and controls. Conclusion: These results suggest that polymorphisms in STAT2, STAT3 and IFN-${\gamma}$ genes are not likely to be strong predictors of cervical cancer in Han women in southern China.

• Asian-Australasian Journal of Animal Sciences
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• 제18권4호
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• pp.483-489
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• 2005

To understand the molecular mechanisms that regulate intramuscular fat deposition and its release, cDNA clones expressed in adipose tissues of Korean cattle were identified by differential screening from adipose tissue cDNA library. By partial nucleotide sequencing of 486 clones and a search for sequence similarity in NCBI nucleotide databases, 245 clones revealed unique clones. By a functional grouping of the clones, 14% of the clones were categorized to metabolism and enzyme-related group (stearoyl CoA desaturase, lactate dehydrogenase, fatty acid synthase, ATP citrate lyase, lipoprotein lipase, acetyl CoA synthetase, etc), and 6% to signal transduction/cell cycle-related group (C/EBP, cAMP-regulated phosphoprotein, calmodulin, cyclin G1, cyclin H, etc), and 4% to cytoskeleton and extracellular matrix components (vimentin, ankyrin 2, gelosin, syntenin, talin, prefoldin 5). The obtained 245 clones will be useful to study lipid metabolism and signal transduction pathway in adipose tissues and to study obesity in human. Some clones were subjected to full-sequencing containing open reading frame. The cDNA clone of bovine homolog of human prefoldin 5 gene had a total length of 959 nucleotides coding for 139 amino acids. Comparison of the deduced amino acid sequences of bovine prefoldin 5 with those of human and mouse showed over 95% identity. The cDNA clone of bovine homolog of human ubiquitin-like/S30 ribosomal fusion protein gene had a total length of 484 nucleotides coding for 133 amino acids. Comparison of the deduced amino acid sequences of bovine ubiquitin-like/S30 ribosomal fusion protein gene with those of human, rat and mouse showed over 97% identity. The cDNA clone of bovine homolog of human proteolipid protein 2 mRNA had a total length of 928 nucleotides coding for 152 amino acids. Comparison of the deduced amino acid sequences of bovine proteolipid protein 2 with those of human and mouse showed 87.5% similarity. The cDNA clone of bovine homolog of rat thymosin beta 4 had a total length of 602 nucleotides coding for 44 amino acids. Comparison of the deduced amino acid sequences of bovine thymosin beta 4 gene with those of human, mouse and rat showed 93.1% similarity. The cDNA clone of bovine homolog of human myotrophin mRNA had a total length of 790 nucleotides coding for 118 amino acids. Comparison of the deduced amino acid sequences of bovine myotrophin gene with those of human, mouse and rat showed 83.9% similarity. The functional role of these clones in adipose tissues needs to be established.

• 생명과학회지
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• 제20권5호
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• pp.655-661
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• 2010

흑색종세포주 SK-MEL-2에서 레티노이드에 의한 GD3합성효소(hST8Sia I)의 발현억제기작을 규명하게 위하여 hST8Sia I의 프로모터 활성을 조사해 본 결과 -1146에서 -646영역에서 레티노이드에 의한 활성억제를 나타내었다. 또한 부위특이적 변이와 ChIP분석은 -731에서 -722영역에 위치한 전사인자NF-kB 결합부위가 hST8Sia I의 레티노이드에 의한 활성억제에 중요하게 관여하고 있음을 나타내었다. 이러한 발현 억제는 PKC/ERK 신호전달경로를 통하여 일어난다는 것을 신호전달경로 저해제를 이용한 RT-PCR과 프로모터 활성조사에 의해 규명하였다.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.83-83
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• 2003

The lutropin/choriogonadotropin receptor (LHR) is a member of the rhodopsin-like subfamily of G protein coupled receptor (GPCRs), that has been shown to mediate the internalization of its two naturally occurring agonist, lutropin and choriogonadotropin (CG). The clustered agonist-receptor complex is internalized by a dynamin-dependent pathway and traverses the endosomal compartment without agonist dissociation Dissociation of the agonist-receptor complex occurs in the lysosomes, where both the agonist and receptor are degrade. Recently, constitutively activating mutations of the receptor have been identified that are associated with familial male-precocious puberty (FMPP). A FMPP is a form of sexual precocious puberty in boys in which testosterone levels are elevated independent of changes in luteinizing hormone-releasing hormone and serum luteinizing hormone levels, We have now analyzed two naturally occurring, constitutively active mutants of the human LHR. These mutations were introduced into the rat LHR (rLHR) and are designated L435R and D556Y. Cells expressing rLHR-D556Y bind human choriogonadotropin (hCG) with normal affinity, exhibit a 25-fold increase in basal cAMP and respond to hCG with a normal increase in cAMP accumulation. Cells expressing rLHR-L435R also bind hCG with normal affinity, exhibit a 47-fold increase in basal cAMP, and do not respond to hCG with a further increase in cAMP accumulation. This mutation enhances the internalization of the free and agonist-occupied receptors ~2- and ~17- fold, respectively We conclude that the state of activation of the rLHR can modulate its basal and/or agonist-stimulated internalization. Since the internalization of hCG is involved in the termination of hCG actions, we suggest that the lack of responsiveness detected in cells expressing rLHR-L435R is due to the fast rate of internalization of the bound hCG. The finding that membranes expressing rLHR-L435R respond to hCG with an increase in adenylyl cyclase activity supports this suggestion. Autonomous Leydig cell activity in FMPP is caused by a constitutively activating LH/CGR.