Natural Compounds Induce Klotho Promoter Activity And Klotho Promoter Analysis

Klotho is a newly identified aging-suppressor gene.It has been shown that Klotho mutant mice show a number of aging-like phenotypes,such as short lifespan, infertility,ectopic calcification,arteriosclerosis,skin atrophy,osteoporosis and emphysema.Moreover,over-expression of Klotho in mice results in the extension of lifespan.Therefore,screening and identification of the activator of Klotho promoter could be used for the development of anti-aging agents.In the previous study,a stable cell line was established by transfecting HEK293 cell line with a vector containg a 1.6kb Klotho promoter fragment linked with luciferase reporter gene.The cells were treated with 0.0006%hydrogen peroxide for 6 hours to establish an oxidative stress cell model.To identify activators of Klotho gene promoter,we have screened a natural product library derived from Traditional Chinese Medicine using the cell model.Our results showed that a small molecule compound,Marmesin,which was purified from Notopterygium incisum could activate Klotho promoter.In this paper,we verified the activity of the compound and found that its EC50 value was 1.60μg/ml(6.6μM).An aging animal model has been established by injection of D-galactose (500mg/kg·day) into ICR mice for 6 weeks.The aging mice were given two dose of Marmesin(5mg/kg·day and 10mg/kg·day) by IP injection for 30 days.The expression levels of Klotho and other molecular marker of aging were measured.The results showed that the SOD level of erythrocyte was significantly decreased in the aging mice,while MDA level of serum was significantly increased after 10mg/kg·day and 5mg/kg·day Marmesin treatment.In addition,serum phosphorus and serum calcium level of aging mice were significantly increased,while 10mg/kg·day Marmesin treatment could reduce phosphorus level in serum.Surprisingly,the compound treatment did not affect the SOD and MDA level of liver and Klotho gene expression in kidney.In order to study the expression and regulation of Klotho gene and identify the response area of Marmesin on the promoter,we cloned a 4.2Kb promoter from 5'-flanking of human Klotho gene.TESS analysis showed that human Klotho gene promoter was lack of TATA BOX.There are multiple SP1 sites in its transcription initiation site.We have made series deletions of the promoter and fused the different promoter fragments into a luciferase reporter gene plasmid.The promoter-luciferase constructs were transfected into human renal embryonic cell line HEK293.The results showed that the core promoter region of Klotho gene was the area from transcription starting point to -400bp.Further analysis suggested that there could be a silencer in the region of -127bp to -243bp.A remote enhancer was identified in the region of -1.6Kb to -3Kb.The regulatory element responds to Marmesin is located in region from transcription starting point to -127bp.Interestingly,we found that treatment of cells with 150μM paraquat,a oxidative stress inducer,for 6 hours could significantly increase the Klotho gene promoter activity,and its response element was located in region from transcription starting point to -127bp.This study suggested that oxidative stress could induce Klotho gene promoter activity.