Endocrine Abstracts

Understanding human pancreatic beta cell differentiation provides safe, normal pathways by which to manipulate stem cells for the ambitious treatment of Type 1 diabetes (1-3). The transcription factor Neurogenin 3 (Ngn3) is a transient, yet critical regulator of this process, committing progenitor cells to an endocrine cell fate (4). However, the downstream consequences of NGN3 expression remain incompletely understood.

To identify new target genes of NGN3, we have developed a model of tetracycline-inducible expression (Tet-On system, BD Biosciences) in the human PANC1 pancreatic ductal carcinoma cell line. NGN3 is expressed only in the presence of the tetracycline derivative, Doxycycline (Dox). We screened over 200 stably transfected clones by transient transfection and RNA dot blot to identify the few that induced NGN3 only in the presence of Dox. In four, NGN3 transcripts were detected by Northern analysis after 2 h stimulation, with maximal detection after ~24 h and absence ~24 h after Dox removal. Transactivation of NEUROD1, a known target of NGN3, was detected by RT-PCR and luciferase reporter analysis after 48 h. For the best clone, microarray analysis (Amersham Codelink System) of NGN3 induction reassuringly had no genetic consequences for the vast majority of genes (>16,000/20,000). However, several hundred demonstrated modified gene expression of a similar level to NGN3. 43 up-regulated genes are expressed in pancreatic islets or insulinoma cells, generating some excellent candidate targets of NGN3 action (including known genes such as SOX4, NEUROD1, PAX4). We are currently validating these novel targets with a view to studying their function directly during beta cell differentiation within the human early fetal pancreas.

1. K Piper et al., Mech Dev 116, 223 (2002).

2. K Piper et al., Diabetologia 45, 1045 (2002).

3. K Piper et al., J Endocrinol 181, 11 (2004).

4. H Edlund, Nat Rev Genet 3, 524 (2002).