| Organic anion transporting polypeptides(Human: OATPs,animals: Oatps)are important cell membrane transporters.They are widely distributed in different tissues and organs such as the liver,kidney,small intestine,testis,and blood brain barrier.Substrates of OATPs include large amount of endogenous and exogenous substances such as steroids,bile salts,thyroid hormones,drugs,and toxins.Therefore,it is well accepted that OATPs play a critical role in absorption,distribution,and excretion of various drugs.Nuclear receptors are a class of ligand activated transcription factors,which play an important role in the growth and development of organisms.PXR(Pregnane X receptor),CAR(Constitutive androgen receptor),LXR(Liver X receptor)and FXR(Farnesoid X receptor)are important members of the nuclear receptor family and are widely distributed in vivo.They are involved in regulation of many metabolizing enzymes,transporters and signaling pathways after ligand activation.It has been reported that many OATPs are regulated by nuclear receptors in human and model animals,but studies on Oatps transcription regulation in food animals are still limited.OATP1A2 is an important member of the OATP family,it can transport a wide variety of drugs,and thus affect the accumulation of drugs within the cell.Previous studies in our laboratory cloned an isoform of OATP1A2 from pig and named it swine Oatp1a2(sOATP1a2).We found that sOATP1a2 is highly expressed in the liver and hence may play an important role in drug absorption,distribution and excretion within the pig as well.Since OATPs/Oatps can transport wide variety of drugs including antibiotics,a better understand of the regulatory mechanisms Oatp1a2 will enable us make good use of this transporter to increase drug absorption efficiency and reduce drug residual.In the current study,we tried to evaluate the regulatory effect of four main nuclear receptors PXR,CAR,LXR and FXR on pig Oatp1a2.The results obtained are as follows.(1)Computer analysis of the sequence located 2147 bp upstream of the Slco1a2 transcription initiation site revealed 5 possible nuclear receptor target sites,namely IR2(-2030 bp ~-2017 bp),DR1(-1704 bp ~-1692 bp),ER1(-1476 bp ~-1464 bp),DR8(-1409 bp~-1390 bp)and DR3(-653 bp ~-639 bp);(2)Co-transfection of LXR or FXR in HepG2 and LLC-PK1 cells both significantly increased activity of the luciferase constructs carrying the 2147 bp upstream sequence,with a more pronounced effect by FXR.Co-transfection with PXR and CAR,however,showed no significant effect;(3)Cells that co-transfected with different nuclear receptor and luciferase construct were treated with corresponding nuclear receptor agonists,and it was found that agonists of LXR and FXR significantly increased the activity of luciferase;while agonists of PXR and CAR had no significant effect on the luciferase expression;(4)A series of luciferase constructs with individual nuclear receptors target site sequence were then generated.It was found that the constructs with IR2(-2030 bp ~-2017 bp)and DR8(-1409 bp ~-1390 bp)exhibited strong response to FXR agonist GW4064,suggesting that FXR may regulate Slco1a2 through these two Site.The IR2 construct also showed a strong response to LXR agonist GW3965,suggesting the regulation of Slco1a2 by LXR might be achieved through this site as well;(5)By mutating the highly conserved bases at IR2 and DR8,it was found that the mutants lost its responses toward GW4064.Preliminary studies with electrophoretic mobility shift assay(EMSA)showed that IR2 may directly interact with FXR;(6)When LLC-PK1 cells that do not normally express Slco1a2 were transfected FXR and treated with GW4064,a marginal but significant up-regulation of porcine Slco1a2 expression was observed. |
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