Transcription Factor Glis3, a Novel Critical Player in the Regulation of Pancreatic β-Cell Development and Insulin Gene Expression

Abstract

In this study, we report that the Krüppel-like zinc finger transcription factor Gli-similar 3 (Glis3) is induced during the secondary transition of pancreatic development, a stage of cell lineage specification and extensive patterning, and that Glis3 zf/zf mutant mice develop neonatal diabetes, evidenced by hyperglycemia and hypoinsulinemia. The Glis3 zf/zf mutant mouse pancreas shows a dramatic loss of ␤ and ␦ cells, contrasting a smaller relative loss of ␣, PP, and cells. In addition, Glis3 zf/zf mutant mice develop ductal cysts, while no significant changes were observed in acini. Gene expression profiling and immunofluorescent staining demonstrated that the expression of pancreatic hormones and several transcription factors important in endocrine cell development, including Ngn3, MafA, and Pdx1, were significantly decreased in the developing pancreata of Glis3 zf/zf mutant mice. The population of pancreatic progenitors appears not to be greatly affected in Glis3 zf/zf mutant mice; however, the number of neurogenin 3 (Ngn3)-positive endocrine cell progenitors is significantly reduced. Our study indicates that Glis3 plays a key role in cell lineage specification, particularly in the development of mature pancreatic ␤ cells. In addition, we provide evidence that Glis3 regulates insulin gene expression through two Glis-binding sites in its proximal promoter, indicating that Glis3 also regulates ␤-cell function.

Modulation of the Transactivation Function and Stability of Krüppel-like Zinc Finger Protein Gli-similar 3 (Glis3) by Suppressor of Fused

“…1A). To determine whether the observed pattern of regulation was ␤-cell-specific, the experiment was repeated in HEK293 cells, which were used previously to demonstrate the ability of Glis3 to activate a (GlisBS) 6 -driven reporter (9). The pattern of transcriptional activation by various Glis3 mutants observed in HEK293 cells was qualitatively very similar to that observed in INS-1 cells (Fig.…”

“…pCMV-Myc-SUFU was kindly provided by Rune Toftgard (Karolinska Institutet, Huddinge, Sweden) (22). The mIP(Ϫ696)-Luc luciferase reporter construct was described previously (6). pM-Glis3 was generated by inserting the full-length coding region of Glis3 into the pM expression vector (Clontech) following PCR amplification.…”

“…Generation of Reporter and Expression Constructs-The generation of p3xFlag-CMV-Glis1, p3xFlag-CMV-Glis2, p3xFlag-CMV-Glis3, p3xFlagCMV-Glis3⌬N302, p3xFlag-CMVGlis2⌬N58, p3xFlag-CMV-Glis2⌬N160, and p3xFlag-CMVGlis2⌬C169 was described previously (6,9,20,21). The plasmids p3xFlag-CMV-Glis3⌬N34, -65, -155, -288, -295, -319, -333, -355, and -388 and p3xFlag-CMV-Glis3⌬C389 were generated by PCR amplification of the respective fragments and inserting them into the EcoRI and BamHI restriction sites of the p3xFlag-CMV10 expression vector (Sigma).…”

“…Genetic aberrations in the GLIS3 gene have been linked to the development of neonatal diabetes and congenital hypothyroidism, polycystic kidney disease, and liver fibrosis, as well as type 1 and type 2 diabetes (11)(12)(13)(14)(15)(16). Mice with a disrupted Glis3 function develop neonatal diabetes, hypothyroidism, and polycystic kidney disease (6,7,17). Recently, Glis3 has been shown to positively regulate transcription of the human proinsulin (INS) and rodent insulin 2 (Ins2) genes in pancreatic ␤-cells (7,8).…”

“…Glis3 has been implicated in the regulation of several physiological processes, including osteoblast differentiation, pancreatic development, and the maintenance of normal renal functions (1,5,6,10,11). Genetic aberrations in the GLIS3 gene have been linked to the development of neonatal diabetes and congenital hypothyroidism, polycystic kidney disease, and liver fibrosis, as well as type 1 and type 2 diabetes (11)(12)(13)(14)(15)(16).…”