Cooperation Of PDX-1 And MKL-1/Myocardin Promotes Insulin Transcription

Diabetes is regarded as one of the four major noncommunicable diseases by WHO, and has been a serious threat to human life and health. Thus, studies on the transcription regulation of insulin gene is a hot in the field of diabetes treatment. The transcription factor, pancreatic duodenal homeobox-1 (PDX-1) plays an important role in pancreas development, beta cell differentiation and maintaining mature β-cell function. PDX-1 can interact with a variety of transcription factors to promote insulin gene transcription and expression, such as MafA, ATF2 and Beta2, etc. It has been reported that PDX-1 and SRF could synergistically promote transcription of rat insulin promoter.MKL-1/Myocardin are strong co-factors of SRF. In this study, how PDX-1 and MKL-1/Myocardin synergistically promoted the transcription of insulin gene were investigated.Firstly, the protein and protein interactions between PDX-1 and MKL-1 in vivo were confirmed by Co-IP technology. Further, the relative structural domains of PDX-1 (transactivation domain, Pro-rich domain, homodomain and C-terminal domain)were inserted to pGEX-6p-3 vector, and then several PDX-1-GST fusion proteins were expressed. MKL-1 proteins were expressed by eukaryotic expression system, and tnen GST pull-down experiments were carried out. Results indicated that the N domain of PDX-1 (1-79aa) could interact with MKL-1.To father studied whether PDX-1 and MKL-1 could promote the insulin gene transcription activation together, the luciferase report gene expression plasmid containing tne rat insulin Ⅱ promoter. (RIP Ⅱ)were constructed, and then the ability of PDX-1 and MKL-1 to promote RIP Ⅱ transcription activation were detected by luciferase saaays. The results showed PDX-1 and MKL-1 could enhance the activity of RIP II together. The sequence analysis indicated that RIP II promoter contains multiple CArG Boxes and A Boxes. In order to.verify which site PDX-1 and MKL-1 binded to and promoted insulin gene transcription activation, the plasmids containing mutated CArG Box or A Box, and the truncated RIP Ⅱ luciferase reporter gene plasmids missing the distal CArG Box, A3 Box and Al Box were constructed. Then luciferase assays were performed. Results showed that the cooperation of PDX-1 and MKL-1 depended on the combination between the distal CArG Box and A Boxes.Futhermore, in this study, the cooperation of PDX-1 and Myocardin,which belongs to the family of MKL-1, was investigated, too. The data determined that, Myocardin could interact with PDX-1 to activate insulin transcription, and the action mainly depended on CArG Box.