(651b) A Discrete Modeling Approach to Identify the Signaling Pathways That Leads to the Induction of Activating Transcription Factor 4 (ATF4) and cAMP Responsive Element Binding Protein (CREB) by Palmitate in HepG2 Cells

The activating transcription factor 4 (ATF4) is induced by stress signals and plays an important role in regulating cell death and survival. We observed that a saturated free fatty acid, palmitate, induces ATF4 in human hepatocellular carcinoma (HepG2) cells, which may suggest a novel signaling pathway induced by palmitate. It is well recognized that inhibiting eukaryotic translation initiation factor 2α (eIF2α) by PKR-like ER kinase (PERK) enables ATF4 translation, however, the transcriptional regulation of ATF4 is not clearly understood. In this study, we observed that another ATF/CREB (activating transcription factor/cAMP response element binding protein) family transcription factor CREB is also activated by palmitate. Promoter analysis revealed that ATF4 contains a putative CRE binding site, therefore we hypothesize that activation of CREB may be contributing to ATF4 upregulation. Indeed, overexpressing CREB increased ATF4 mRNA levels and silencing CREB reduced ATF4 levels. Further, there is positive feedback from ATF4 to CREB activation. Such observations, together with previous studies on the pathways induced upon palmitate stimulation, suggest that the dynamic behavior of the signaling pathway through ATF4 is tuned by a variety of feedback circuits. Therefore, we applied a discrete dynamic modeling approach, which provides an in silico framework, to integrate and assess the contributions of the various components in the pathway on the dynamic behavior of the signaling process. We combine experiments with model simulations to delineate the essential pathways that are involved in the regulation of ATF4 and CREB activation. Our modeling and experimental results demonstrate how an integrated view of the dynamic process can better characterize the dynamic regulatory mechanisms at play, and thereby enhance our understanding of the palmitate induced signaling aberrations in cellular regulatory networks.