A cell-based assay system for activators of the environmental cell stress response.

Improved health span and lifespan extension in a wide phylogenetic range of species is associated with the induction of the environmental cell stress response through a signalling pathway regulated by the transcription factor Nrf2. Phytochemicals which stimulate this response may form part of therapeutic interventions which stimulate endogenous cytoprotective mechanisms, thereby delaying the onset of age-related diseases and promoting healthy ageing in humans. In order to identify compounds that activate the Nrf2 pathway, a cell-based reporter system was established in HepG2 cells using a luciferase reporter gene under the control of the Nqo1 promoter. Sulforaphane, an isothiocyanate derived from cruciferous vegetables and a known activator of the Nrf2 pathway, was used to validate the reporter system. The transfected cell line HepG2 C1 was subsequently used to screen natural product libraries. Five compounds were identified as activating the bioluminescent reporter by greater than 5-fold. The two most potent compounds, MBC20 and MBC37, were further characterised and shown to stimulate endogenous cytoprotective gene and protein expression. The bioluminescent reporter system will allow rapid, in vitro identification of novel compounds that have the potential to improve health span through activation of the environmental stress response.

LIF-dependent survival of embryonic stem cells is regulated by a novel palmitoylated Gab1 signalling protein.

The cytokine leukaemia inhibitory factor (LIF) promotes self-renewal of mouse embryonic stem cells (ESCs) through activation of the transcription factor Stat3. However, the contribution of other ancillary pathways stimulated by LIF in ESCs, such as the MAPK and PI3K pathways, is less well understood. We show here that naive-type mouse ESCs express high levels of a novel effector of the MAPK and PI3K pathways. This effector is an isoform of the Gab1 (Grb2-associated binder protein 1) adaptor protein that lacks the N-terminal pleckstrin homology (PH) membrane-binding domain. Although not essential for rapid unrestricted growth of ESCs under optimal conditions, the novel Gab1 variant (Gab1ß) is required for LIF-mediated cell survival under conditions of limited nutrient availability. This enhanced survival is absolutely dependent upon a latent palmitoylation site that targets Gab1ß directly to ESC membranes. These results show that constitutive association of Gab1 with membranes through a novel mechanism promotes LIF-dependent survival of murine ESCs in nutrient-poor conditions.