Abstract
BackgroundHeterozygous paired box6 (Pax6) mutations lead to abnormal glucose metabolism in mice older than 6 months as well as in human beings. Our previous study found that Pax6 deficiency caused down-expression of prohormone convertase 1/3 (Pcsk1), resulting in defective proinsulin processing. As a protein cleaving enzyme, in addition to its expression, the activity of PC1/3 is closely related to its function. We therefore hypothesize that Pax6 mutation alters the activity of PC1/3, which affects proinsulin processing.Methodology/Principal FindingsUsing quantitative RT-PCR, western blot and enzyme assay, we found that PC1/3 C-terminal cleavage and its activity were compromised in Pax6 R266Stop mutant mice, and the expression of Pcsk1n, a potent inhibitor of PC1/3, was elevated by Pax6 deficiency in the mutant mice and MIN6 cells. We confirmed the effect of proSAAS, the protein encoded by Pcsk1n, on PC1/3 C-terminal cleavage and its activity by Pcsk1n RNAi in MIN6 cells. Furthermore, by luciferase-reporter analysis, chromatin immunoprecipitation, and electrophoretic mobility shift assay, we revealed that Pax6 bound to Pcsk1n promoter and directly down-regulated its expression. Finally, by co-transfecting Pax6 siRNA with Pcsk1n siRNA, we showed that Pax6 knock-down inhibited proinsulin processing and that this effect could be rescued by proSAAS down-regulation. These findings confirm that Pax6 regulates proinsulin processing partially through proSAAS-mediated PC1/3 processing and activity.Conclusions/SignificanceCollectively, the above experiments demonstrate that Pax6 can directly down-regulate Pcsk1n expression, which negatively affects PC1/3 C-terminal cleavage and activity and subsequently participates in proinsulin processing. We identified proSAAS as a novel down-regulated target of Pax6 in the regulation of glucose metabolism. This study also provides a complete molecular mechanism for the Pax6 deficiency-caused diabetes.
Highlights
Paired box 6 (Pax6) is a transcription factor and a member of the paired-box gene family [1,2]
The results indicate that the mutant mice have defective prohormone convertase 1/3 (PC1/3) processing in paired box6 (Pax6) mutated islets
In addition to investigating PC1/3 C-terminal cleavage, we investigated the activity of PC1/3 in the islets of the Pax6 mutant mice
Summary
Paired box 6 (Pax6) is a transcription factor and a member of the paired-box gene family [1,2]. We found that Pax directly regulated the expression of prohormone convertase 1/3 (PC1/3) encoded by the gene Pcsk, a serine protease essential for proinsulin processing. The down-regulation of Pcsk led to defective proinsulin processing and abnormal glucose metabolism in Pax mutant mice and in PAX6-deficient patients as well [10]. Mice with a disruption of Pcsk exhibited growth retardation and several hormone precursors processing defects including proinsulin [11,12] This conclusion was further supported by a human pedigree study, which reported that the PCSK1 mutation exhibited phenotypes including, but not limited to severe obesity, abnormal glucose homeostasis, elevated plasma proinsulin, low insulin level [13]. Our previous study found that Pax deficiency caused down-expression of prohormone convertase 1/3 (Pcsk1), resulting in defective proinsulin processing. We hypothesize that Pax mutation alters the activity of PC1/3, which affects proinsulin processing
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