Pax6 Directly Down-regulates Pcsk1n Expression Thereby Regulating PC1/3 Dependent Proinsulin Processing

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2012 Oct 12

PMID 23056534

Citations 16

Authors

Ting Liu

Yanxia Zhao

Na Tang

Ruopeng Feng

Xiaolong Yang

Nicole Lu

Jinhua Wen

Lingsong Li

Affiliations

Soon will be listed here.

Abstract

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.

Methodology/principal Findings: 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/significance: 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.

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References

Boudreault A, Gauthier D, Rondeau N, Savaria D, Seidah N, Chretien M . Molecular characterization, enzymatic analysis, and purification of murine proprotein convertase-1/3 (PC1/PC3) secreted from recombinant baculovirus-infected insect cells. Protein Expr Purif. 1999; 14(3):353-66. DOI: 10.1006/prep.1998.0964. View

Simpson T, Price D . Pax6; a pleiotropic player in development. Bioessays. 2002; 24(11):1041-51. DOI: 10.1002/bies.10174. View

Wen J, Chen Y, Song S, Ding J, Gao Y, Hu Q . Paired box 6 (PAX6) regulates glucose metabolism via proinsulin processing mediated by prohormone convertase 1/3 (PC1/3). Diabetologia. 2008; 52(3):504-13. DOI: 10.1007/s00125-008-1210-x. View

Clark J, Gebhart G, Gonder J, Keeling M, Kohn D . Special Report: The 1996 Guide for the Care and Use of Laboratory Animals. ILAR J. 1997; 38(1):41-48. DOI: 10.1093/ilar.38.1.41. View

Wei S, Feng Y, Che F, Pan H, Mzhavia N, Devi L . Obesity and diabetes in transgenic mice expressing proSAAS. J Endocrinol. 2004; 180(3):357-68. DOI: 10.1677/joe.0.1800357. View

Zhu X, Zhou A, Dey A, Norrbom C, Carroll R, Zhang C . Disruption of PC1/3 expression in mice causes dwarfism and multiple neuroendocrine peptide processing defects. Proc Natl Acad Sci U S A. 2002; 99(16):10293-8. PMC: 124907. DOI: 10.1073/pnas.162352599. View

Jackson R, Creemers J, Farooqi I, Raffin-Sanson M, Varro A, Dockray G . Small-intestinal dysfunction accompanies the complex endocrinopathy of human proprotein convertase 1 deficiency. J Clin Invest. 2003; 112(10):1550-60. PMC: 259128. DOI: 10.1172/JCI18784. View

Lamango N, Zhu X, Lindberg I . Purification and enzymatic characterization of recombinant prohormone convertase 2: stabilization of activity by 21 kDa 7B2. Arch Biochem Biophys. 1996; 330(2):238-50. DOI: 10.1006/abbi.1996.0249. View

Fortenberry Y, Hwang J, Apletalina E, Lindberg I . Functional characterization of ProSAAS: similarities and differences with 7B2. J Biol Chem. 2001; 277(7):5175-86. DOI: 10.1074/jbc.M104531200. View

10.

Quiring R, Walldorf U, Kloter U, Gehring W . Homology of the eyeless gene of Drosophila to the Small eye gene in mice and Aniridia in humans. Science. 1994; 265(5173):785-9. DOI: 10.1126/science.7914031. View

11.

Sander M, Neubuser A, Kalamaras J, Ee H, Martin G, German M . Genetic analysis reveals that PAX6 is required for normal transcription of pancreatic hormone genes and islet development. Genes Dev. 1997; 11(13):1662-73. DOI: 10.1101/gad.11.13.1662. View

12.

Kozmik Z, Czerny T, Busslinger M . Alternatively spliced insertions in the paired domain restrict the DNA sequence specificity of Pax6 and Pax8. EMBO J. 1998; 16(22):6793-803. PMC: 1170283. DOI: 10.1093/emboj/16.22.6793. View

13.

Jang E, Goldman J . Pax6 expression is sufficient to induce a neurogenic fate in glial progenitors of the neonatal subventricular zone. PLoS One. 2011; 6(6):e20894. PMC: 3117849. DOI: 10.1371/journal.pone.0020894. View

14.

Qian Y, Devi L, Mzhavia N, Munzer S, Seidah N, Fricker L . The C-terminal region of proSAAS is a potent inhibitor of prohormone convertase 1. J Biol Chem. 2000; 275(31):23596-601. DOI: 10.1074/jbc.M001583200. View

15.

Katz L, Gosmain Y, Marthinet E, Philippe J . Pax6 regulates the proglucagon processing enzyme PC2 and its chaperone 7B2. Mol Cell Biol. 2009; 29(8):2322-34. PMC: 2663301. DOI: 10.1128/MCB.01543-08. View

16.

Cameron A, Fortenberry Y, Lindberg I . The SAAS granin exhibits structural and functional homology to 7B2 and contains a highly potent hexapeptide inhibitor of PC1. FEBS Lett. 2000; 473(2):135-8. DOI: 10.1016/s0014-5793(00)01511-8. View

17.

Mishra R, Gorlov I, Chao L, Singh S, Saunders G . PAX6, paired domain influences sequence recognition by the homeodomain. J Biol Chem. 2002; 277(51):49488-94. DOI: 10.1074/jbc.M206478200. View

18.

Hohmeier H, Newgard C . Cell lines derived from pancreatic islets. Mol Cell Endocrinol. 2004; 228(1-2):121-8. DOI: 10.1016/j.mce.2004.04.017. View

19.

Zhu X, Orci L, Carroll R, Norrbom C, Ravazzola M, Steiner D . Severe block in processing of proinsulin to insulin accompanied by elevation of des-64,65 proinsulin intermediates in islets of mice lacking prohormone convertase 1/3. Proc Natl Acad Sci U S A. 2002; 99(16):10299-304. PMC: 124908. DOI: 10.1073/pnas.162352799. View

20.

Yasuda T, Kajimoto Y, Fujitani Y, Watada H, Yamamoto S, Watarai T . PAX6 mutation as a genetic factor common to aniridia and glucose intolerance. Diabetes. 2002; 51(1):224-30. DOI: 10.2337/diabetes.51.1.224. View