Wild-type PAX4 and PAX4 R192H proteins were expressed in vitro and the cell compartmentalization of each protein was examined after transfection of the plasmid constructs into ¦ÂTC3 cells followed by Western-blot analysis. The plasmid containing wild-type PAX4 or PAX4 R192H was co-transfected into ¦ÂTC3 and ¦ÁTC-1.9 cells with insulin or glucagon promoter-reporter construct. Transcriptional repression activities were then determined by dual-luciferase reporter assay.
Wild-type PAX4 and PAX4 R192H, which were found to be equally expressed in vitro and transfection systems, were present in the nuclear compartment. Transcriptional repressor activities of PAX4 R192H on human insulin and glucagon promoters were reduced when they were compared with those of wild-type PAX4.
These results suggested that PAX4 R192H polymorphism generated a protein with defect in transcriptional repressor activities on its target genes, which may lead to ¦Â-cell dysfunction associated with MODY and early onset-age of T2D as reported in our previous study.