The International Journal of Biochemistry & Cell Biology 44 (2012) 72–83 Contents lists available at SciVerse ScienceDirect The International Journal of Biochemistry & Cell Biology jo ur nal homep ag e: www.elsevier.com/locate/biocel SOX9 regulates endocrine cell differentiation during human fetal pancreas development Erin McDonald a , Jinming Li a,b , Mansa Krishnamurthy a , George F. Fellows d , Cynthia G. Goodyer e , Rennian Wang a,b,c,∗ a Children’s Health Research Institute, University of Western Ontario, London, ON N6C 2V5, Canada b Physiology & Pharmacology, University of Western Ontario, London, ON N6C 2V5, Canada c Medicine, University of Western Ontario, London, ON N6C 2V5, Canada d Obstetrics and Gynecology, University of Western Ontario, London, ON N6C 2V5, Canada e Department of Pediatrics, McGill University, Montreal, QC H3Z 2Z3, Canada a r t i c l e i n f o Article history: Received 29 April 2011 Received in revised form 7 September 2011 Accepted 22 September 2011 Available online 29 September 2011 Keywords: Human fetal pancreas Human islet-epithelial cells Islet transcription factors Akt/GSK3 pathway Beta- and alpha-cell differentiation a b s t r a c t The transition of pancreatic progenitor cells to mature endocrine cells is regulated by the sequential activation and interaction of several transcription factors. In mice, the transcription factor Sox9 has been shown to support endocrine cell differentiation. However, the functional role of SOX9 during pancreas development in the human has yet to be determined. The present study was to characterize SOX9 expres- sion during human fetal pancreas development and examine its functional role by transfection with SOX9 siRNA or SOX9 expression vectors. Here we report that SOX9 was most frequently expressed in PDX1 + cells (60–83%) and least in mature endocrine cells (<1–14%). The proliferation of SOX9 + cells was sig- nificantly higher at 8-10weeks than at 14–21weeks (p < 0.05) or 20–21weeks (p < 0.01). SOX9 frequently co-localized with FOXA2, NGN3 and transcription factors linked to NGN3 (NKX2.2, NKX6.1, PAX6). siRNA knockdown of SOX9 significantly decreased islet-epithelial cell proliferation, NGN3, NKX6.1, PAX6 and INS mRNA levels and the number of NGN3 + and insulin + cells (p < 0.05) while increasing GCG mRNA and glucagon + cells (p < 0.05). Examination of SOX9 associated signaling pathways revealed a decrease in phospho-Akt (p < 0.01), phospho-GSK3 (p < 0.01) and cyclin D1 (p < 0.01) with a decrease in nuclear -catenin + (p < 0.05) cells following SOX9 siRNA knockdown. In contrast, over-expression of SOX9 sig- nificantly increased the number of islet cells proliferating, NGN3, NKX6.1, PAX6 and INS mRNA levels, the phospho-Akt/GSK3 cascade and the number of insulin + cells. Our results demonstrated that SOX9 is important for the expression of NGN3 and molecular markers of endocrine cell differentiation in the human fetal pancreas. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction One major cause of the progression of diabetes is a decline in pancreatic beta-cell mass and function. Therapeutic strategies aimed at repopulating insulin-producing cells show great poten- tial for restoring normal blood glucose levels and, thus, extensive research efforts are focused on developing methods to differ- entiate progenitor cells into beta-cells (Hao et al., 2006; Jiang et al., 2007; Meier et al., 2005) and to maintain their viability and function (Bonner-Weir and Sharma, 2002; Heit and Kim, 2004). However, such an undertaking requires a thorough analysis of tran- scription factors that control pancreatic organogenesis and tissue ∗ Corresponding author at: Victoria Research Laboratories, Room A5-140, 800 Commissioners Road East, London, Ontario N6C 2V5, Canada. Tel.: +1 519 685 8500x55098; fax: +1 519 685 8186. E-mail address: rwang@uwo.ca (R. Wang). maintenance. One important player in this complex process is the SOX, or sex-determining region on Y box, family of transcription factors which is involved in cell fate determination and the devel- opment of several tissues during embryogenesis (Akiyama et al., 2002; De Santa Barbara et al., 1998; van de Wetering et al., 1993; Wegner, 1999). These transcription factors share a high mobil- ity group (HMG) DNA binding domain which alone fulfills the functions of DNA binding and bending, protein interactions and nuclear import or export (Wilson and Koopman, 2002). Several members of this family are under investigation for their role in murine pancreas development (Lioubinski et al., 2003; McDonald et al., 2009), including Sox4, Sox6, Sox9 and Sox17 with regards to endoderm specification (Kanai-Azuma et al., 2002), maintain- ing pancreatic progenitor cell status and supporting endocrine cell differentiation (Lynn et al., 2007; Seymour et al., 2007; Wilson et al., 2005), beta-cell proliferation and insulin secretion (Iguchi et al., 2005, 2007) and endocrine cell differentiation (Mavropoulos et al., 2005; Wilson et al., 2005). Only SOX9 expression has been 1357-2725/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2011.09.008