Pinoresinol-4,4′-di-O-β-d-glucoside from Valeriana officinalis root stimulates calcium mobilization and chemotactic migration of mouse embryo fibroblasts

Lignans are major constituents of plant extracts and have important pharmacological effects on mammalian cells. Here we showed that pinoresinol-4,4′-di-O-β-d-glucoside (PDG) from Valeriana officinalis induced calcium mobilization and cell migration through the activation of lysophosphatidic acid (LPA) receptor subtypes. Stimulation of mouse embryo fibroblast (MEF) cells with 10 μM PDG resulted in strong stimulation of MEF cell migration and the EC₅₀ was about 2 μM. Pretreatment with pertussis toxin (PTX), an inhibitor of G_i protein, completely blocked PDG-induced cell migration demonstrating that PDG evokes MEF cell migration through the activation of the G_i-coupled receptor. Furthermore, pretreatment of MEF cells with Ki16425 (10 μM), which is a selective antagonist for LPA₁ and LPA₃ receptors, completely blocked PDG-induced cell migration. Likewise, PDG strongly induced calcium mobilization, which was also blocked by Ki16425 in a dose-dependent manner. Prior occupation of the LPA receptor with LPA itself completely blocked PDG-induced calcium mobilization. Finally, PDG-induced MEF cell migration was attenuated by pretreatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor such as LY294002. Cells lacking downstream mediator of PI3K such as Akt1 and Akt2 (DKO cells) showed loss of PDG-induced migration. Re-expression of Akt1 (but not Akt2) completely restored PDG-induced DKO cell migration. Given these results, we conclude that PDG is a strong inducer of cell migration. We suggest that the pharmacological action of PDG may occur through the activation of an LPA receptor whereby activation of PI3K/Akt signaling pathway mediates PDG-induced MEF cell migration.     

Functional interaction between peroxisome proliferator-activated receptors-alpha and Mef-2C on human carnitine palmitoyltransferase 1beta (CPT1beta) gene activation

Muscle-type carnitine palmitoyltransferase 1 (CPT1β) is considered to be the gene that controls fatty acid mitochondrial β-oxidation. A functional peroxisome proliferator-activated receptor (PPAR) responsive element (PPRE) and a myocite-specific (MEF2) site that binds MEF2A and MEF2C in the promoter of this gene had been previously identified. We investigated the roles of the PPRE and the MEF2 binding sites and the potential interaction between PPARα and MEF2C regulating the CPT1β gene promoter. Mutation analysis indicated that the MEF2 site contributed to the activation of the CPT1β promoter by PPAR in C2C12 cells. The reporter construct containing the PPRE and the MEF2C site was synergistically activated by co-expression of PPAR, retinoid X receptor (RXR) and MEF2C in non-muscle cells. Moreover, protein-binding assays demonstrated that MEF2C and PPAR specifically bound to one another in vitro. Also for the synergistic activation of the CPT1β gene promoter by MEF2C and PPARα-RXRα, a precise arrangement of its binding sites was essential.     

Epigenetic regulation of Cebpb activation by pY19-Caveolin-2 at the nuclear periphery in association with the nuclear lamina

Here, we show that Caveolin-2 (Cav-2) is an epigenetic regulator for adipogenesis. Upon adipogenic stimulation, inner nuclear membrane (INM)-targeted pY19-Cav-2 interacted with lamin A/C to disengage the repressed Cebpb promoter from lamin A/C, which facilitated the Cebpb promoter association with lamin B1. Consequently, pY19-Cav-2 recruited lysine demethylase 4b (KDM4b) for demethylation of histone H3 lysine 9 trimethylation (H3K9me3) and histone acetyltransferase GCN5 for acetylation of H3K27, and subsequently RNA polymerase II (Pol II) on Cebpb promoter for epigenetic activation of Cebpb, to initiate adipogenesis. Cav-2 knock-down abrogated the Cebpb activation and blocked the Pparg2 and Cebpa activation. Re-expression of Cav-2 restored Cebpb activation and adipogenesis in Cav-2-deficient preadipocytes. Our data identify a new mechanism by which the epigenetic activation of Cebpb is controlled at the nuclear periphery to promote adipogenesis.