Preliminary Study On Klotho's Transcriptional Regulation

The incidence of aging is a complex mechanism, multiple factors involved in the pathophysiological process. Aging theory, from cells perspective, includes oxidatie stress theory, mitochondrial damage theory, telomerase doctrine and so on; From etiology standpoint, the reasons of aging are mainly the imbalance of gene regulation,disorder of internal environment and the influence of external factors.Gene-regulation theory about aging explains the reasons of aging and mechanisms of age-related disorders mainly from the aspects of promoter's activity, transcriptional factors and methylation modification of genes. Klotho is closely related to aging, and studies reveal that mutation in klotho in mouse causes extensive aging phenotypes including arteriosclerosis, vascular calcifications, soft tissue calcifications, emphysema, hypoactivity, gonadal dysplasia, infertility, skin atrophy, ataxia, hypoglycemia and severe hyperphosphatemia. However, over-expression of klotho in mouse can postphone aging. According to the present researches, the mechanisms of klotho's anti-aging role are characterized by the following aspects. At first, klotho protein can suppress the insulin/ insulin like growth factor-1 signal pathway,which is evolutionarily highly conserved anti-aging mechanism. Secondly, klotho protein,as a co-factor of FGF23,involves in phosphate metabolism. And experiments confirm that high serum phosphate level was related to aging.At last, klotho protein can increase the resistance for oxidant stress, protecting organisms from damages of oxidative stress.In addition, klotho protein can suppress other growth factor signal pathways,regulate immune functions and participate in adipocyte differentiation.The recent studies reveal that klotho takes part in the occurrence and development of aging-related diseases. Our early work revealed the G-395A polymorphism in klotho was associated with arteriosclerosis.What's more,it is responsible for essential hypertension and may be a potential gene regulatory site.The functions of klotho protein are wide and significant, but transcriptional regulation of klotho is unknown. Our concentration is the regulation of klotho. With the completion of human genome project,most genes have been located and recognized,whose functions and transcriptional mechanisms have been found,but some genes remain unkown,including klotho. Klotho gene is located in chromosome 13q12,which is composed of 5 exons and 4 introns. Promoter region of klotho don't include typical TATA- box and CAAT- box, but exist 5 SP1-binding sites. The klotho gene plays a role in phenotypic alterations in various organs,and expression of klotho mRNA is predominantly observed in the kidney. Previous studies observed downregulation of klotho in the kidney in several rat models including the spontaneously hypertensive rat,the deoxycorticosterone acetate-salt hypertensiverat,5/6 nephrectomized rat, non-insulin-dependent diabetes mellitus rat.The downregulation of klotho gene also happened in mouse model for ageing–related renal injury. These suggested that expression of klotho was downregulated by oxidative stress. In addition,there are response elements for Peroxisome Proliferator-activated receptorγ(PPAR-γ)in klotho gene promoter ,and experiments confirmed that PPAR-γsensitizer can upregulate the expression of klotho. However,transcription factors and response elements, as well as the mechanisms of promoter activity's change are unknown and need to be a further study.Researches found that the overexpression of klotho can improve renal function of diabetic nephropathy rats and reduce oxidatie stress damage induced by high sugar.Then, what effect does oxidatie stress have on transcriptional regulation of klotho?Aging-related oxidatie stress theory considers that increased reactive oxygen is related to aging of cell and tissue. The recent studies reveal that reactive oxygen accelerates the aging of cells,which is deduced to activate signal pathway including Akt/PKB and ERK signal pathways to activate aging-related genes so as to effect aging. Reactive oxygen, as a signal regulational factor,can regulate the expression of some aging-related genes,such as p66shc, Sirtuin, FOX03 and klotho.Our concentration is reactive oxygen's effect on klotho promoter's activity.Our experiment takes klotho promoter as a key point, to explore the location of klotho promoter and its activity in different cells.Which is an elementary research about transcriptional regulation of klotho. Furthermore,we make models for oxidatie stress with H₂O₂ to analyse the effect of H₂O₂ on activity of klotho promoter in HeLa cell and the relevance with antioxidant system.The main experimental methods and results:One part: Genomic DNA of human red cell was used as the template, klotho promoter segments with 137,312,498,967bp lengths were amplifed by PCR and then respectively cloned into pGL₃-Basic vectors.The recombinant vectors were transformed into DH5αto screen positive clones, getting 4 recombinant vectors:pGL₃-Basic-KLI,pGL₃-Basic- KLII,pGL₃-Basic-KLIII,pGL₃-Basic-KLIV.These can be tested by double digestion and sequencing. The recombinant vectors respectively with pRL-TK vector were co-transfected into HEK293 and HeLa cells with lipofectamine 2000. The transcriptional activities were analyzed by double luciferase method. The result reveals that the activities of pGL₃-Basic- KL III and pGL₃-Basic-KLIV were markedly higher compared with pGL₃-Basic in HEK293(p<0.01),which suggested the ability to start pGL₃-Basic reporter gene.The activity of pGL₃-Basic-KLIII was notably higher compared with pGL₃-Basic in HeLa cell(p<0.05),however, the activity of pGL₃-Basic-KLIV was significantly decreased compared with pGL₃-Basic- KLIII(p<0.01).Two part:Genomic DNA of human red cell was used as the template, klotho promoter segments with 967 bp lengths were amplifed by PCR and then cloned into pGL₃-Basic vectors.The recombinant vector was transformed into DH5αto screen positive clone, getting pGL₃-Basic -KL.It can be tested trough double digestion and sequencing. The pGL₃- Basic-KL with pRL-TK vector were co-transfected into HeLa cells with lipofectamine 2000. Then 400,600,800 and 1000umol/L concentrations of H₂O₂ is exposed to HeLa cell , known as 1,2,3 and 4 group,taking 0 umol/L H₂O₂ as control group,then observe luciferase value and the activity of T-AOC,CAT and GSH-PX. The result shows that the klotho promoter activities of 2,3 and 4 group were significantly down-regulated compared with control group(p<0.01).The level of klotho promoter activity is positively related with the activities of T-AOC,CAT and GSH-PX(r=0.805,0.812,0.944, P=0.000).Conclusion:One part: The sequence -504～-6 may be a core promoter region;the expression level of pGL₃-Basic-KLIV is different in HEK293 and HeLa cell,and the sequence -973～-504 bp maybe responsible for this difference.Two part: Oxidative stress can down-regulate klotho promoter's activity ,which is positively related with the decrease of oxidative resistance.