U-937 세포에 있어서 세라마이드에 의한 c-jun 유전자 발현의 조절

김원호,김미영,최경희,Kim, Won-Ho,Kim, Mie-Young,Choi, Kyung-Hee 대한약학회 1997 약학회지 Vol.41 No.1

Ceramide has been suggested as an important mediator of the effects of extracellular agonists on cell growth inhibition, differentiation, apoptosis. However the biochemical sign aling mechanism involved in transducing the effects of ceramide on leukemia cell differentiation is still unclear. In these respects, we examined the regulatory effects of ceramide on c-jun gene expression during differentiation. In U-937 cells. ceramide increased c-jun mRNA levels in a time-dependent manner. The half life, of c-jun mRNA was 30 min. In contrast, inhibition of protein synthesis with cycloheximide in the absence, of transcription with actinomycin D increased the half-life of c-jun mRNA in ceramide-treated U-937 cells to more than 90 min. In order to examine whether ceramide-inhibited c-jun gene expression is regulated through ceramide-activated protein phosphatase (CAPP), a direct target for the action of ceramide, okadaic acid were treated to the cells. Okadaic acid inhibited enhancement of c-jun mRNA induced by C2-ceramide in a dose-dependent manner. These results suggested that ceramide increases c-jun mRNA level during differentiation in U-937 cells and regulates the gene expression on posttranscriptional level. In addition, we provide the evidence that CAPP is involved in ceramide-induced c-jun gene expression in U-937 cells.

Ceramide-Mediated Cell Death Was Accompanied with Changes of c-Myc and Rb Protein

Moon,Soon-Ok,Lee,Jin-Woo The Korea Science and Technology Center 1998 BMB Reports Vol.31 No.4

The sphingomyelin cycle and ceramide generation have been recognized as potential growth suppression signals in mammalian cells. Ceramide has been shown to induce differentiation, cell growth arrest, senescence, and apoptosis. Although the intracelluar target for the action of ceramide remains unknown, recent studies have demonstrated the role of cytosolic ceramide-activated protein phosphatase (CAPP). In this study, the cytotoxic effect of C2-ceramide, a synthetic cell-permeable ceramide analog, on HEp-2 cells and the mechanism by which ceramide induces cell death were investigated. The addition of exogenous C2-ceramide resulted in a concentration dependent cell death. Okadaic acid, a potent inhibitor of CAPP, enhanced ceramide-mediated cell death, which suggests that CAPP is not involved in this process. To understand the mechanism of action of ceramide, we studied the relationship between ceramide and c-Myc and pRb which are defined components of cell growth regulation. Western blot analyses revealed that C2-ceramide (10μM) induced c-Myc down-regulation, but there were no significant changes in pRb. However, treatment of okadaic acid (10nM) enhanced c-Myc and pRb down-regulation. Reduction of the amount of c-Myc and pRb occurred during HEp-2 cell death. These results suggest that the cytotoxic effect of ceramide in HEp-2 cells may not be mediated through the action of CAPP and that the downstream target for ceramide is c-Myc and pRb.

c-myc gene expression by ceramide-mediated pathway in U-937 cells

Kim,Mie Young,Choi,Kyung Hee,Kim,Keun Cheol 中央大學校 遺傳工學硏究所 1994 遺傳工學硏究論集 Vol.7 No.1

본 연구에서는 sphingomyelin 가수분해 생성물인 ceramide에 의한 c-myc 유전자 발현 경로를 알아보기 위해 U-937세포에 ceramide를 처리하여 다음과 같은 실험결과를 얻었다. 첫째, U-937 세포에 0.5~6μM의 ceramide를 1시간 처리하면 농도 의존적으로 c-myc 유전자 발현이 감소되었으며 둘째, PKC의 활성 유도제인 500nM을 1시간 처리하였을 때의 c-myc protooncogene 발현이 증가되나 6μM mide를 19시간 전처리하였을 경우 1시간 이내에 감소하였다. 셋째, PMA를 19시간 전처리하면 ceramide에 의한 c-myc 유전자의 발현은 더욱 감소되었다. 이상의 결과로서 PKC 의존적 경로에 위한 c-myc 유전자 발현에 ceramide에 의한 신호 전달 경로가 조절 작용을 나타냄을 알 수 있었다. The present study examines the possibility that ceramide-mediated pathway may serve to regulate the c-myc gene expression by PKC-dependent pathway in U-937 cells. Treatment of U-937 cells with a cell permeable ceramide caused a decrease in c-myc mRNA levels in a dose-dependent manner. A 60% reduction occurred at 3μM. The level of c-myc mRNA was reduced to 76% of control in the cells pretreated with ceramide for 19 hours and then treated with PMA for 1 hour suggesting that ceramide-mediated pathway antagonized the c-myc gene expression by PKC-dependent pathway However, in the combinatory pretreatment of PMA for 19 hours and ceramide for 1 hour c-myc gene expression was drastically reduced, possibly reflecting that PKC-dependent pathway had a synergistic effects on c-myc gene expression by ceramide-mediated pathway.

N-oleoyl-D-erythro-sphingosine-based Analysis of Ceramide by High Performance Liquid Chromatography and Its Application to Determination in Diverse Biological Samples

Lee, Youn-Sun,Choi, Heon-Kyo,Yoo, Jae-Myung,Choi, Kyong-Mi,Lee, Yong-Moon,Oh, Sei-Kwan,Kim, Tack-Joong,Yun, Yeo-Pyo,Hong, Jin-Tae,Okino, Nozomu,Ito, Makoto,Yoo, Hwan-Soo The Korean Society of Toxicogenomics and Toxicopro 2007 Molecular & cellular toxicology Vol.3 No.4

Ceramide is involved in cell death as a lipid mediator of stress responses. In this study, we developed an improved method of ceramide quantification based on added synthetic ceramide and thin layer chromatography (TLC) separation, and applied to biological samples. Lipids were extracted from samples spiked with N-oleoyl-D-erythro-sphingosine ($C_{17}$ ceramide) as an internal standard. Ceramide was resolved by TLC, complexed with fatty-acidfree bovine serum albumin (BSA), and deacylated by ceramidase (CDase). The released sphingosine was derivatized with o-phthalaldehyde (OPA) and measured by high performance liquid chromatography (HPLC). The limit of detection for ceramide was about 1-2 pmol and the lower limit of quantification was 5 pmol. Ceramide recovery was approximately 86-93%. Ceramide concentrations were determined in biological samples including cultured cells, mouse tissues, and mouse and human plasma. TLC separation of ceramide provides HPLC chromatogram with a clean background without any interfering peaks and the enhanced solubility of ceramide by BSAceramide complex leads to the increased deacylation of ceramide. The use of an internal standard for the determination of ceramide concentration in these samples provides an accurate and reproducible analytical method, and this method can be applicable to diverse biological samples.

Ceramide-Mediated Cell Death Was Accompanied with Changes of c-Myc and Rb Protein

Moon, Soon-Ok,Lee, Jin-Woo Korean Society for Biochemistry and Molecular Biol 1998 Journal of biochemistry and molecular biology Vol.31 No.4

The sphingomyelin cycle and ceramide generation have been recognized as potential growth suppression signals in mammalian cells. Ceramide has been shown to induce differentiation, cell growth arrest, senescence, and apoptosis. Although the intracelluar target for the action of ceramide remains unknown, recent studies have demonstrated the role of cytosolic ceramideactivated protein phosphatase(CAPP). In this study, the cytotoxic effect of C2-ceramide, a synthetic cellpermeable ceramide analog, on HEp-2 cells and the mechanism by which ceramide induces cell death were investigated. The addition of exogenous C2-ceramide resulted in a concentration dependent cell death. Okadaic acid, a potent inhibitor of CAPP, enhanced ceramide-mediated cell death, which suggests that CAPP is not involved in this process. To understand the mechanism of action of ceramide, we studied the relationship between ceramide and c-Myc and pRb which are defined components of cell growth regulation. Western blot analyses revealed that C2-ceramide (10${\mu}M$) induced c-Myc down-regulation, but there were no significant changes in pRb. However, treatment of okadaic acid (10 nM) enhanced c-Myc and pRb down-regulation. Reduction of the amount of c-Myc and pRb occurred during HEp-2 cell death. These results suggest that the cytotoxic effect of ceramide in HEp-2 cells may not be mediated through the action of CAPP and that the downstream target for ceramide is c-Myc and pRb.

Stress-induced senescence of dermal papilla cells restored by synthesized ceramide

( Ji Hee Jung ),( Young Jun Woo ),( Ki Min Sohn ),( Jee Hye Oh ),( Kwan Ho Jeong ),( Jung Eun Kim ),( Hoon Kang ) 대한피부과학회 2017 대한피부과학회 학술발표대회집 Vol.69 No.2

Background: Dermal papilla cells (DPCs) are known to regulate the cell senescence via regulation of proliferation and apoptosis. Ceramide is a second messenger of bioactive sphingolipid that mediates a variety of cell functions involved in apoptosis, cell differentiation, proliferation and cellular senescence. However, the effects of ceramide on stress-induced senescence of hair follicles were not well identified yet. Objectives: We aimed to confirm the effects of ceramide on regulating growth factors including ERK/Akt and Wnt/β-catenin pathway in senescence-induced DPCs. Methods: To investigate the ceramide-related pathway, we evaluated how ceramide affects ERK/Akt activation and Wnt/β-catenin signaling in H₂O₂-treated DPCs. Results: We found that Akt phosphorylation in H₂O₂-treated DPCs was decreased by ceramide, whereas ERK phosphorylation was significantly upregulated by ceramide. Wnt5a and β-catenin mRNA levels were increased in ceramide alone group. Interestingly, ceramide upregulated the expression of Wnt10b in H₂O₂-treated DPCs. Conclusion: These results suggest that ceramide may be closely related to the maintenance of hair follicles in senescence environment such as aging or damaging by certain stimuli. Ceramide may be useful for adjunctive material to delay the hair senescence.

신세포암 세포에서 Ceramide의 성장 억제 효과 및 기전에 관한 연구

김청수,홍준혁,박형근,홍지희,정진수,박노정,황온유,박태한 大韓泌尿器科學會 1999 Korean Journal of Urology Vol.40 No.3

실크단백질의 식이 공급이 아토피 피부염 동물 모델 NC/Nga Mice 피부의 세라마이드 함량 및 관련인자 발현에 미치는 영향

박경호,최영심,김현애,이광길,여주홍,정도현,김성한,조윤희,Park, Kyung-Ho,Choi, Young-Sim,Kim, Hyun-Ae,Lee, Kwang-Gill,Yeo, Joo-Hong,Jung, Do-Hyun,Kim, Sung-Han,Cho, Yun-Hi 한국식품영양과학회 2007 한국식품영양과학회지 Vol.36 No.5

본 연구에서는 아토피 피부염 동물 모델인 NC/Nga mice에 실크단백질 sericin과 fibroin을 식이 공급 후 피부의 세라마이드 함량 및 관련인자 발현 변화를 정상대조군인 BALB/c mice 및 아토피 피부염 대조군과 비교 분석하였다. 세라마이드 함량은 아토피 대조군인 CA군이 정상대조군 C군보다 현저히 낮았으나, 정제된 건조 분말을 별다른 처리 없이 그대로 실크단백질 sericin을 식이공급한 S군은 정상대조군인 C군 이상의 수준으로 높였으며, 실크단백질 fibroin(F군)은 세라마이드 함량을 정상대조군 수준이상으로 올려주지는 못하였다. 세라마이드 합성효소인 SPT의 mRNA 및 protein 발현은 아토피 대조군인 CA군은 정상대조군인 C군보다 높았으나, 실크단백질 공급군인 S군 및 F군은 모두 정상대조군 C군보다 현저히 낮았다. 반면, 세라마이드 분해효소인 ceramidase의 mRNA 및 protein 발현은 아토피 대조군인 CA군은 정상대조군인 C군보다 높았으나, 실크단백질을 식이섭취한 S군 및 F군에서는 정상대조군인 C군과 유사한 수준으로 CA군에 비해서는 유의적으로 낮았다. 결론적으로 실크단백질 sericin의 10주간 식이섭취는 아토피 피부염 동물모델 NC/Nga mice 표피의 세라마이드 함량을 정상대조군 수준 이상으로 증가시켰으며, 궁극적으로 표피의 장벽기능을 정상대조군의 수준으로 변화시켰다. 이는 serine이 다량 함유되어있는 실크단백질 sericin을 공급에 의해 증가되었는데, serine은 그 자체로서 보습기능을 하여 표피의 장벽기능을 보완하였기 때문에 SPT에 의한 생합성은 불필요하였으며 또한, ceramidase에 의한 세라마이드 분해를 정상수준이하로 억제하여 세라마이드의 생성보다는 분해억제에 의한 것으로 여겨진다. Ceramide rich intercellular lipid lamellae are thought to be particularly important in maintaining the structural integrity of epidermal barrier. Ceramide is synthesized de novo by serine palmitoyltransferase (SPT) phospholipid intermediates, serine and palmitic acid persist within the stratum corneum. The ceramide which is synthesized is degraded with fatty acid and sphingosine by degradative enzyme ceramidase. The depletion of ceramide in stratum corneum was reported in the atopic dermatitis. As an effort to search for the dietary source for improving the level of ceramide in epidermis, the dietary effects of various-typed silk protein were compared. Seventy male NC/Nga mice, an animal model of atopic dermatitis, were divided into seven groups: group CA as an atopic control with control diet, group S: 1% crude sericin diet, group F: 1% crude fibroin diet, group PS : peptide pattern of sericin(Mw 5000), group PF: peptide pattern of fibroin (Mw 1500), group AS: manufactured the same as amino acid profile of sericin and group AF: manufactured the same as amino acid profile of fibroin. Ten male BALB/c mice were served as group C (control group) control diet. All mice were fed on diet and water ad libitum for 10 weeks. Dry skin condition was established in group CA as ceramide content was decreased. Despite a marked decrease of mRNA and prorein expression of SPT, enzyme do novo synthesis, ceramide content of group S was dramatically increased by inhibiting the mRNA and protein expression of degradative enzyme ceramidase. However, dietary supplementation of crude silk fibroin protein (group F) and in other groups that were supplemented with either amino acid or peptide type of sericin or fibroin did not increase the level of ceramide. Together, our data demonstrate that dietary supplementation of crude sericin is more effective at improving ceramide level in epidemis of NC/Nga mice.

The effect of ceramide-based essence cream for the damaged hair shaft

( Young Jun Woo ),( Kwan Ho Jeong ),( Jee Hye Oh ),( Hye Ree Park ),( Jung Eun Kim ),( Hoon Kang ) 대한피부과학회 2018 대한피부과학회 학술발표대회집 Vol.70 No.2

Background: Ceramides exist inside the hair cuticle to act as cement, keeping the hair intact. Hair cuticles are often thought as densely packed shingles on a roof, and ceramides are the glue that keeps the shingles together Objectives: We aimed to investigate the protective effect of ceramide formulated essence cream on the damaged hair. Methods: Hair samples were divided into normal (n=4) and damaged groups (n=6). Contents of cholesterol and free fatty acid (linoleic acid and oleic acid) in hair samples were measured by gas chromatography-mass spectrometry (GC-MS) analysis. Also, we selected virgin hair samples and let them chemically and frictionally damaged. Each damaged hair was treated with five different ceramide essence creams (vehicle, stearyl, oleyl, stearyl-cholesterol, and stearyl-linoleic acid). Results: Contents of cholesterol (688 ug/g) and linoleic acid (133 ug/g) in damaged hairs were lower than normal hairs (cholesterol (1025 ug/g) and linoleic acid (200 ug/g)) by GC-MS analysis. In contrast, the content of cholesterol (2089 ug/g) of hair samples wasincreased in ceramide-treated normal hair groups (1025 ug/g). To measure the surface state of hair, we performed scanning electron microscope for ultrastructure of damaged hair cuticle fraction. Cuticle layer of damaged hair surface was improved after the treatment with ceramide essence cream. Conclusion: Ceramide-based essence cream improved damaged hair, especially, ceramide with stearyl and ceramide with stearyl-cholesterol

세라마이드 함유 에토좀의 물성과 피부흡수

현통일(Tong-Il Hyeon),윤경섭(Kyung-Sup Yoon) 한국응용과학기술학회 (구.한국유화학회) 2021 한국응용과학기술학회지 Vol.38 No.3

바이오틴을 피부에 전달하기 위해 고압균질기를 사용하여 바이오틴과 세라마이드를 모두 함유하는 에토좀에 대하여 연구하였다. 바이오틴이 주된 성분으로 사용되었으며, 세라마이드 NP는 인지질 이중층의 지지체로 활용되었다. 바이오틴은 수용성 내부에 포획되었고, 세라마이드 NP는 에토좀의 이중층에 흡착되었다. 세라마이드 NP를 함유한 에토좀의 물성을 살펴보면 소포체의 크기는 80∼130 nm, 다분산지수는 0.09∼0.16, 제타 전위는 -40∼-49 mV로 측정되었다. 세라마이드 NP가 없는 소포 체의 크기는 124.80±1.46 nm, 다분산지수와 제타전위는 각각 0.088±0.018과 -45.48±1.27 mV이었 다. 따라서 세라마이드 NP가 함유된 에토좀은 세라마이드 NP가 없는 에토좀에 비해 소포체의 물리적 특성이 개선됨을 알 수 있었다. 세라마이드 NP를 함유한 에토좀의 피부흡수율은 12시간 후 6.13∼ 14.98%이었으며, 반면에 세라마이드 NP가 없는 에토좀의 피부흡수율은 7.08%이었다. 결론적으로 세라 마이드 NP를 함유한 에토좀은 피부흡수 효율을 향상시킬뿐만 아니라 소포체의 안정성에도 긍정적인 영향을 미쳤다. In order to delivery biotin to skin, ethosomes containing both biotin and ceramide were researched by using high pressure homogenizer. Biotin was utilized as a drug and ceramide NP was utilized as a supporter of bilayer. The biotin was entrapped in aqueous core, while ceramide NP was packed in the bilayer of the ethosomes. Looking at the physical properties of vesicles containing ceramide NP, the sized was 80∼130 nm, the polydispersity index was 0.09∼ 0.16, and the zeta potential was -40∼-49 mV. In vesicles without ceramide NP, the size was 124.80±1.46 nm, and the zeta potential and polydispersity index were -45.48±1.27 mV and 0.088±0.018, respectively. Therefore, the ethosome with ceramide NP has improved physical properties of vesicles compared to the ethosome without ceramide NP. Skin absorption rates of ethosomes with ceramide NP were 6.13∼14.98%, while skin absorption rate of ethosome without ceramide NP was 7.08% at 12 h. In conclusion, ethosomes containing ceramide NP not only improved the skin absorption efficiency, but had also a positive effect on the stability of vesicles.