Differentiation Potential of Pancreatic Fibroblastoid Cells/Stellate Cells: Effects of Peroxisome Proliferator-Activated Receptor Gamma Ligands
Figure 7
Gel shift analysis of PPARγ. (a) Electrophoretic mobility shift analyses (EMSAs) in hPFC showed DNA binding of PPARγ under control conditions, in the absence of ligand and in response to ciglitazone (3.3 μM) stimulation using a PPARγ consensus sequence. Supershift analyses with anti-PPARγ antibodies were included to show the specificity of PPARγ binding. Binding to a retinoid X receptor (RXR) consensus sequence was visible without ciglitazone treatment, but more pronounced after ligand binding to PPARγ. Supershift analysis revealed that PPARγ was present in the complex binding to RXR. (b) Analysis of PPARγ binding in rat PFC1 cells. Two different concentrations of ciglitazone were used to show induction of dimer formation and binding to the RXR consensus sequence. A clear induction is visible with the PPARγ probe already at the lower concentration. Supershift analysis showed that principally the lower of the two bands contained PPARγ, while the upper band remained in place. (c) Analysis of PPARγ binding in transformed HPF-T cells. Different stimulators were used for differentiation purposes. Analysis of stimulation with ciglitazone (3.3 μM) was compared to 15Δ-prostaglandin J2 (PGJ2 at 5 μM), next to all trans retinoic acid (ATRA) and 9-cis-retinoic acid (9cisRA). The lower PPARγ band was induced probing with RXR by ciglitazone and PGJ2, but ATRA and 9cisRA inductors for retinoid receptors did not stimulate PPARγ binding to this consensus sequence. Using the consensus sequence of rat acyl coenzyme A oxidase, the signal was considerably weaker, but still induction of receptor binding was induced by specific ligands, while RXR binding to this probe appeared reduced.
