cAMP-Coupled riboflavin trafficking in placental trophoblasts: a dynamic and ordered process.

Dietary intake of free riboflavin (vitamin B2; RF)1 or following release from the coenzyme forms, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) determines the nutritional status of this essential nutrient in humans. Physiological deficiency of RF accounts for clinical manifestations of growth retardation, anemia, cardiovascular disease, and neuro-degenerative disorders (1, 2). Despite the critical role of flavin analogs in normal cellular metabolic processes, little is known about the mechanism and regulation of the vitamin B2 absorptive process. Investigative approaches based on biochemical characterization and two-dimensional fluorescence microscopy have identified a putative receptor-mediated component to be responsible, at least in part, for RF uptake in human enterocytes and trophoblasts. High ligand-binding affinity (nanomolar range), temperature- and ion-dependence, and a saturable process that can be inhibited by structurally related ligands are the salient features that describe this system (3-5). Analogous to iron recruitment and absorption, the proposed RF entry mechanism involves initial recognition by specific binding proteins that chaperone the hydrophilic ligand across membrane barriers within vesicles (6). However, the identity of these proteins and other components that facilitate the absorption of this essential nutrient remains elusive. Consequently, elucidating the driving mechanisms that contribute to the cellular homeostasis of this vitamin has generated considerable interest from the standpoint of nutritional biology and fetal growth and development, with potential application in targeted drug design and delivery (7, 8). Internalization of extracellular nutrients in eukaryotes via receptor-mediated endocytosis employs complex cell machinery in a sequential and regulated manner. The co-existence of abundant intracellular structural and signaling networks offers several possibilities for receptor-associated ligands. Most water-soluble vitamins are fated to metabolic trapping, a term that describes enzymatic processing of the molecule to forms that are spatially restricted within the cell (9). However, alternative translocation routes, including late endosomal or lysosomal degradation, membrane recycling, or even transcytosis (in polarized epithelial systems) remain a distinct possibility (10). Such receptor-ligand interactions also occur under extensive cellular regulation either via an intrinsic mechanism such as kinase or phosphatase activity or via generation of signaling cascades that include second messenger regulation of integrated pathways (11, 12). Huang and colleagues have demonstrated that the RF internalization process is affected by modulation of the cyclic nucleotide second messengers, cAMP and cGMP, as well as by protein kinase A, G and calcium-calmodulin pathways (4). The objective of the current study was to evaluate the fate of internalized RF over time by following its intracellular sorting using the human placental trophoblast cell line, BeWo (13) and define the regulatory role of cyclic AMP in this process. The enhanced nutritional requirements of the fetus (14-17) and the presence of a high affinity RF system (4) justify the use of the selected model that was compared with another human polarized epithelial system, Caco-2 cells with nanomolar affinity for RF (3). Time- and concentration-dependent fractionation analyses of the localized ligand in endosomes, lysosomes, Golgi, and mitochondria were compared with confocal laser scanning microscopy studies examining time-lapse 3-D colocalization between internalized rhodamine-labeled RF (Rd-RF) (18, 19) and immunolabeled organelles. Trafficking profiles for RF were compared with those determined for transferrin (TF), a receptor-mediated endocytic marker, (20-22), and, for taurocholic acid (TCA) that does not follow a vesicular pathway (23, 24). In addition, cyclic AMP assays were included to determine regulation of the downstream events of cellular RF translocation upon its binding to the proposed RF-receptor. Overall, the study for the first time shows that RF, like TF, accumulates mainly within endosomes but differs from TF in the extent of lysosomal distribution. The present study also suggests a possible recycling mechanism that contributes to RF homeostasis and regulation via the cAMP signaling pathway in the trophoblast model.