Pax6 Controls the Expression of Critical Genes Involved in Pancreatic α Cell Differentiation and Function*

Eric Marthinet,Audrey Guérardel,Liora S. Katz,Yvan Gosmain,Claire Cheyssac,Aline Mamin,Jacques Philippe,Karim Bouzakri

doi:10.1074/jbc.m110.147215

Eric Marthinet, Audrey Guérardel + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m110.147215

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Abstract

The paired box homeodomain Pax6 is crucial for endocrine cell development and function and plays an essential role in glucose homeostasis. Indeed, mutations of Pax6 are associated with diabetic phenotype. Importantly, homozygous mutant mice for Pax6 are characterized by markedly decreased β and δ cells and absent α cells. To better understand the critical role that Pax6 exerts in glucagon-producing cells, we developed a model of primary rat α cells. To study the transcriptional network of Pax6 in adult and differentiated α cells, we generated Pax6-deficient primary rat α cells and glucagon-producing cells, using either specific siRNA or cells expressing constitutively a dominant-negative form of Pax6. In primary rat α cells, we confirm that Pax6 controls the transcription of the Proglucagon and processing enzyme PC2 genes and identify three new target genes coding for MafB, cMaf, and NeuroD1/Beta2, which are all critical for Glucagon gene transcription and α cell differentiation. Furthermore, we demonstrate that Pax6 directly binds and activates the promoter region of the three genes through specific binding sites and that constitutive expression of a dominant-negative form of Pax6 in glucagon-producing cells (InR1G9) inhibits the activities of the promoters. Finally our results suggest that the critical role of Pax6 action on α cell differentiation is independent of those of Arx and Foxa2, two transcription factors that are necessary for α cell development. We conclude that Pax6 is critical for α cell function and differentiation through the transcriptional control of key genes involved in glucagon gene transcription, proglucagon processing, and α cell differentiation.

Highlights

Complementary to ␤ cell dysfunction, it has been well established that ␣ cell dysregulation contributes to the maintenance of fasting and postprandial hyperglycemia in type 2 dia
Conditional inactivation of Pax6 in the mouse pancreas is characterized by a diabetic phenotype with hyperglycemia and hypoinsulinemia reflecting an essential role of Pax6 in the regulation of glucose homeostasis and endocrine cell function [9]
Inasmuch as the presence of glucagon is the most obvious evidence for differentiation of ␣ cells, we investigated whether Pax6 could regulate genes coding for transcription factors involved in glucagon gene transcription

Summary

EXPERIMENTAL PROCEDURES

Islets Isolation—Briefly, islets of Langerhans were isolated by collagenase digestion of pancreases from male adult SpragueDawley rats followed by Ficoll purification using a modification of previously described procedures [28, 29]. ␤ Cells were separated from non-␤ cells by autofluorescence-activated sorting using a FACStar-Plus cell sorter (BD Bioscience), as previously described (29 –31). 16 h after isolation, enriched ␣ cells were cultured on extracellular matrix-coated plates derived from 804G cells (Laminin-5-rich extracellular matrix, 804G-ECM) in DMEM containing 10% fetal calf serum (FCS), 11.2 mM D-Glucose, 2 mM L-Glutamine and antibiotics [32]. Cell Culture—The glucagon producing cell ␣TC1.9 cells were grown in DMEM (Invitrogen) supplemented with 3 g/liter glucose, 10% fetal calf serum, 2 mM glutamine, 100 units/ml of penicillin, and 100 ␮g/ml of streptomycin. The siRNA named siPax – 614 and siPax6 –1007 recognize segments 614/638 and 1007/1031 of the Pax mRNA sequence. Primary adherent enriched ␣ cells (about 40,000 per condition) were transfected with 100 nM of the mixed Pax siRNAs using 1 ␮l of Lipofectamine 2000 (Invitrogen) in 200 ␮l of serum-free medium.

The signal was detected with Super

RESULTS

DISCUSSION