Pax6 directly down-regulates Pcsk1n expression thereby regulating PC1/3 dependent proinsulin processing.
BACKGROUND
Heterozygous 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.


RESULTS
Using 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
Collectively, 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.
Peking University Stem Cell Research Center, Department of Cell Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China.
Ting
T
Liu
Yanxia
Y
Zhao
Na
N
Tang
Ruopeng
R
Feng
Xiaolong
X
Yang
Nicole
N
Lu
Jinhua
J
Wen
Lingsong
L
Li
D000818:Animals; D015536:Down-Regulation; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D009479:Neuropeptides; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D011384:Proinsulin / Q000378:metabolism*; D045664:Proprotein Convertase 1 / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D034741:RNA, Small Interfering / Q000235:genetics; D012097:Repressor Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
D005136:Eye Proteins; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; D009479:Neuropeptides; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; C112832:Pcsk1n protein, mouse; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D011384:Proinsulin; D045664:Proprotein Convertase 1