Expression And Intracellular Effects Of Alternative Splicing Of Organic Solute Transporter Alpha (OSTα)

Objectives 1 To detect tissue expression profiles of the splice variant,OSTα2 in different animals(rabbits,mice,and rats),and human;To detect expression changes of OSTα and OSTα2 in the case of cholestasis or bile acid load,and abnormal lipid metabolism caused by obesity and high-fat diet.2 To detect the effect of OSTα2 on the interaction and membrane localization of OSTα and OSTβ.Methods 1 PCR and semi-nested PCR were used to analyze the expression of OSTα and OSTα2 in the liver,ileum,and kidneys of 10-week-old male C57BL/6 mice and SD rats;Real-time quantitative PCR was used to detect the expression changes of OSTα and OSTα2 in mouse ileum in the case of bile duct ligation or oral bile acid load,and abnormal lipid metabolism caused by obesity and high-fat diet.2 The coding sequences of mouse OSTα,OSTα2,and OSTβ were amplified by PCR and cloned into the expression vector pc DNA3.1(-).The coding sequences of the genes were cloned into the vectors p Bi FC-VC155 and p Bi FC-VN155 for Bi FC analysis and cloned into p EGFPN2(Clonetech)to construct the GFP fusion protein.Analysis of protein interactions and membrane localization using Bi FC and GFP fusion proteins.Results 1 PCR qualitative analysis showed that OSTα2 was expressed in almost all the tissues we detected.The ileal RNA was extracted from each group of mice.Real-time PCR analysis showed that: after bile duct ligation(BDL),the expression of OSTα,OSTα2,and OSTβ was significantly decreased in the BDL 3 days(BDL3d)group compared to control,and that of OSTβ was also decreased in the BDL7 d group.The expression of OSTα was significantly increased after treatment with taurocholate acid(TCA)for 10 days and 20 days.The expression levels of OSTα2 and OSTβ were also significantly elevated in the TCA10 d group;there was no significant change in the cholestyramine(CY)treated group;m RNA expression of OSTα and OSTα2 in the ileum of congenital obesity db/db mice was significantly increased,compared with the control db/m mice;Real-time quantitative PCR results showed that in the nutrition obesity mice caused by high fat diet feeding for 3 months,the expression of OSTα,but not OSTα2,in the ileum was significantly higher than that of control.2 The results of Bi FC analysis and GFP fusion protein analysis showed that the green fluorescence of OSTα and OSTβ co-expression was located on the cell membrane;the homodimer formed by OSTα2 expressed alone in the cytoplasm.The green fluorescence of OSTα2 and OSTβ co-expression was located in the cytoplasm.And the addition of OSTα2 may translocate the heterodimer of OSTα and OSTβ to the cytoplasm.Conclusions 1 The experimental results show that the bile duct ligation decreases the m RNA expression of OSTα,OSTα2 and OSTβ.Treatment with taurocholate acid increases the expression of OSTα,OSTα2 and OSTβ.2 OSTα2 interferes with the formation of OSTα-Ostβ complex,subcellular localization and function by competing with OSTα for binding to Ostβ,showing a dominant negative effect.