The Transcription Factor Rfx3 Regulates β-Cell Differentiation, Function, and Glucokinase Expression

• Published in: Diabetes (New York, N.Y.) Vol. 59; no. 7; pp. 1674 - 1685
• Main Authors: AIT-LOUNIS, Aouatef, BONAL, Claire, SEGUIN-ESTEVEZ, Queralt, SCHMID, Christoph D, BUCHER, Philipp, HERRERA, Pedro L, DURAND, Bénédicte, MEDA, Paolo, REITH, Walter
• Format: Journal Article
• Language: English
• Published: Alexandria, VA American Diabetes Association 01.07.2010
• Subjects: Analysis of Variance; Animals; Biochemistry, Molecular Biology; Biological and medical sciences; Cell Differentiation; Cell Differentiation - physiology; Diabetes. Impaired glucose tolerance; DNA-Binding Proteins; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Endocrine pancreas. Apud cells (diseases); Endocrinopathies; Etiopathogenesis. Screening. Investigations. Target tissue resistance; Fluorescent Antibody Technique; Genetic aspects; Glucokinase; Glucokinase - genetics; Glucokinase - metabolism; Insulin; Insulin - genetics; Insulin - metabolism; Insulin-Secreting Cells; Insulin-Secreting Cells - metabolism; Islet Studies; Islets of Langerhans; Islets of Langerhans - metabolism; Life Sciences; Medical sciences; Mice; Mice, Knockout; Physiological aspects; Promoter Regions, Genetic; Regulatory Factor X Transcription Factors; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Transcription Factors; Transcription Factors - genetics; Transcription Factors - metabolism; Switzerland; Endocrinopathy; Enzyme; Transferases; Diabetes mellitus; Glucokinase; Langerhans islet; β Cell; Cell differentiation; Transcription factor; Endocrine pancreas
• DOI: 10.2337/db09-0986
• ISSN: 0012-1797

Pancreatic islets of perinatal mice lacking the transcription factor Rfx3 exhibit a marked reduction in insulin-producing beta-cells. The objective of this work was to unravel the cellular and molecular mechanisms underlying this deficiency. Immunofluorescence studies and quantitative RT-PCR experiments were used to study the emergence of insulin-positive cells, the expression of transcription factors implicated in the differentiation of beta-cells from endocrine progenitors, and the expression of mature beta-cell markers during development in Rfx3(-/-) and pancreas-specific Rfx3-knockout mice. RNA interference experiments were performed to document the consequences of downregulating Rfx3 expression in Min6 beta-cells. Quantitative chromatin immunoprecipitation (ChIP), ChIP sequencing, and bandshift experiments were used to identify Rfx3 target genes. Reduced development of insulin-positive cells in Rfx3(-/-) mice was not due to deficiencies in endocrine progenitors or beta-lineage specification, but reflected the accumulation of insulin-positive beta-cell precursors and defective beta-cells exhibiting reduced insulin, Glut-2, and Gck expression. Similar incompletely differentiated beta-cells developed in pancreas-specific Rfx3-deficient embryos. Defective beta-cells lacking Glut-2 and Gck expression dominate in Rfx3-deficent adults, leading to glucose intolerance. Attenuated Glut-2 and glucokinase expression, and impaired glucose-stimulated insulin secretion, were also induced by RNA interference-mediated inhibition of Rfx3 expression in Min6 cells. Finally, Rfx3 was found to bind in Min6 cells and human islets to two well-known regulatory sequences, Pal-1 and Pal-2, in the neuroendocrine promoter of the glucokinase gene. Our results show that Rfx3 is required for the differentiation and function of mature beta-cells and regulates the beta-cell promoter of the glucokinase gene.