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Characterization of the endocytic pathways regulating riboflavin (vitamin B2) absorption and trafficking in human epithelial cells

Foraker, Amy Beth
http://rave.ohiolink.edu/etdc/view?acc_num=osu1172865566

Abstract Details

2007, Doctor of Philosophy, Ohio State University, Pharmacy.
Drug bioavailability is greatly compromised by the body’s innate defense mechanisms that result in low membrane permeability of hydrophilic drugs and macromolecules, reduced cellular retention by efflux transporters, and metabolic degradation by a multitude of enzymes. These factors, combined with underlying genetic aberrations and pathologies, drastically reduce drug bioavailability and, in certain instances, increase drug-related toxicity. To address many of these challenges, scientists have devised ways to target therapeutics into cells by coupling drugs directly (i.e., prodrug) or indirectly (e.g., polymer-mediated carriers) to natural ligands that are recognized by membrane receptors, and consequently gain cellular entry through receptor-mediated endocytosis (RME). Although various receptors have been exploited to improve drug delivery, several receptor mechanisms remain to be defined. One RME target that has gained attention for its therapeutic potential in contraception, breast- and liver cancers involves the riboflavin (vitamin B2) RME machinery. Our laboratory recently revealed riboflavin absorption to be regulated, in part, by clathrin-dependent RME (CME) in human epithelia. However, the identity of proteins involved in this process, and an understanding of the intracellular distribution of absorbed B2 remain to be established. The work presented in this dissertation addresses some of these areas of question using a three-tiered approach involving subcellular fractionation of [3H]-B2 dosed cells, 3D fluorescence colocalization analyses of rhodamine-labeled B2 and immunostained endocytic markers, and RNA interference of the endocytic scission enzyme, dynamin 2 GTPase. Herein, we reveal dynamic enrichment of internalized riboflavin to CME endosomes, and to a lesser extent to the Golgi and mitochondria in human placental trophoblasts and enterocytes (Chapter 2). Furthermore, absorbed riboflavin is shown to be negatively coupled to cAMP levels. In Chapter 3, ~ 80% dynamin 2 protein knockdown causes ~ 40% reduced B2-RME, and 2-fold higher B2 localization at the plasma membrane of placental trophoblasts. Lastly, fluorescence colocalization assays exploring the involvement of clathrin-independent caveolae-mediated RME in B2 internalization show minimal B2 enrichment to caveolar endosomes (Chapter 4). Overall, our data demonstrate a dynamic subcellular B2 trafficking itinerary largely associated with CME, and dynamin 2 is the first endocytic protein identified to regulate B2 cellular entry in humans.
Peter Swaan (Advisor)
187 p.
riboflavin; receptor-mediated endocytosis; clathrin; dynamin 2 GTPase; caveolin 1; human epithelia

Recommended Citations

Citations

  • Foraker, A. B. (2007). Characterization of the endocytic pathways regulating riboflavin (vitamin B2) absorption and trafficking in human epithelial cells [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1172865566

    APA Style (7th edition)

  • Foraker, Amy. Characterization of the endocytic pathways regulating riboflavin (vitamin B2) absorption and trafficking in human epithelial cells. 2007. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1172865566.

    MLA Style (8th edition)

  • Foraker, Amy. "Characterization of the endocytic pathways regulating riboflavin (vitamin B2) absorption and trafficking in human epithelial cells." Doctoral dissertation, Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=osu1172865566

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.