Cell-based assay system for high-throughput screening of anti-photo-aging agents in fibroblast transfectants.
The matricellular protein CCN1 is significantly elevated in acutely ultraviolet-irradiated human skin and negatively regulates collagen homeostasis by suppressing collagen synthesis and increasing collagen degradation. In this study, we established a stable cell line, termed CCN1-GFs, by transfection of the pAcGFP1-1-CCN1 promoter plasmid and examined its usefulness as a cell-based assay system for screening anti-aging ingredients. The promoter of the reporter plasmid pAcGFP1-1-CCN1 promoter was transfected into NIH3T3 cells using the Lipofectamine reagent. G418-resistant cells were selected and further cloned. To confirm whether AcGFP1-1-CCN1 promoter plasmid recombined in the NIH3T3 cells, the level of AcGFP1-1-CCN1 was measured by PCR analysis. To determine if NIH3T3 cells expressed the gene encoding green fluorescence protein in a CCN1 promoter-dependent manner, the reporter enzyme activities were assayed using a fluorimeter and flow cytometer. To determine if CCN1 inhibitor, which was selected through this system, exerted a direct effect on the downstream signal, mRNA expression of collagen1 and MMP1A was checked by using real-time PCR. UVB irradiation of CCN1-GFs resulted in increased CCN1 promoter activity. Treatment with retinoic acid, a CCN1 inhibitor, inhibited UV-induced CCN1 promoter activity. Subsequent use of this assay system to screen anti-aging ingredients revealed that CCN1-GFs treated with sclareol showed decreased levels of UVB-induced CCN1 expression. Sclareol attenuated UVB-induced photo-aging by an increase in collagen synthesis and decrease in MMP-1 activity.
Biospectrum Life Science Institute, Sangdaewon-Dong, Seongnam City, Gyeonggi Do, 442-13, Republic of Korea.
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S
Lee
Biospectrum Life Science Institute, Sangdaewon-Dong, Seongnam City, Gyeonggi Do, 442-13, Republic of Korea.
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S
Shin
Biospectrum Life Science Institute, Sangdaewon-Dong, Seongnam City, Gyeonggi Do, 442-13, Republic of Korea. bioso@biospectrum.com.|Biospectrum Life Science Institute, Seongnam City, Gyeonggi Do, 462-807, Repubic of Korea. bioso@biospectrum.com.
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http://orcid.org/0000-0002-5594-4645
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Jung
Biospectrum Life Science Institute, Sangdaewon-Dong, Seongnam City, Gyeonggi Do, 442-13, Republic of Korea.
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D
Park
Anti-photoaging; CCN1 promoter; Cell-based assay; Sclareol; Transfected stable cell