h1

%0 Thesis
%A Spötter, Dorette Christel Else Monika
%T Der Retinoidmetabolismus in HaCaT-Zellen
%C Aachen
%I Publikationsserver der RWTH Aachen University
%M RWTH-CONV-122929
%P IV, 115 S. : Ill., graph. Darst.
%D 2003
%Z Aachen, Techn. Hochsch., Diss., 2003
%X We investigated the metabolism of retinoids in the HaCaT keratinocyte cell line. Vitamin A (Retinol) and its naturally occurring and synthetic derivatives, collectively referred to as retinoids, exert profound effects on several essential life processes, such as vision, reproduction, embryonic development, cell growth, and differentiation. There is also evidence that retinoids are effective in the treatment of a variety of diseases, such as acne and psoriasis. The efficacy of retinoids is mainly influenced by their metabolic pathway. Whereas numerous studies have been performed on the family of retinoid receptors, a relatively large gap in knowledge exists as to how Retinol is metabolized to its active metabolite Retinoic Acid. HaCaT keratinocytes were used in our study as an example of a human non-malignant keratinocyte cell line. This immortal cell line is well characterized and is frequently used as a paradigm for skin keratinocytes in vitro because of its highly preserved differentiation capacity. To investigate the metabolism of retinoids, we used a highly specific RP-HPLC method. We added Retinol, Retinal, and the Retinoid Acid isomers all-trans-RA, 9-cis-RA, and 13-cis-RA, to HaCaT cells in culture and analyzed the concentration of these retinoids, their isomers and their 4-hydroxylated metabolites after a period of 0, 24, 48, 72, 96, and 120 hours of incubation. In addition, we performed RT-PCR with specific primers for enzymes, binding proteins, and receptors known to be involved in the retinoid signaling pathways. Our main finding was the incapability of the HaCaT cells to dehydrogenate Retinol to Retinal. In contrast, if Retinal is added, the cells produce large amounts of Retinoic Acid and also a significant amount of Retinol. In normal human keratinocytes, Retinol is metabolized to Retinal by the enzyme Retinol- Dehydrogenase (RolDH). In our study, there was no detectable mRNA expression of RolDH by RT-PCR. In addition, there was no mRNA induction of the binding protein CRBP I, whose main function is to bind Retinol and to present it to enzymes for further metabolism. On the other hand, mRNA expressions of Retinal-Dehydrogenase (RalDH) and the corresponding binding protein CRBP II were present. RT-PCR also showed an mRNA expression of the proteins CYP 1A1, CYP 1A2, CRABP I, CRABP II and 9-cis-ROLDH. No induction, however, of hLRAT, AHR or PPAR were detectable. With Retinoic Acid as a source, the HaCaT cells showed significant 4-hydroxylation. A comparison of the three different Retinoic Acids isomers suggests that atRA is the most stable isomer and 13-cis-RA is the major origin for the 4-hydroxylated metabolites. Comparison with normal human keratinocytes - which are capable of dehydrogenation Retinol to Retinal and showing a reverse reaction into Retinol if Retinal is used as a source - showed that HaCaT cells were incapable of dehydrogenate Retinol to Retinal.
%F PUB:(DE-HGF)11
%9 Dissertation / PhD Thesis
%R 10.18154/RWTH-CONV-122929
%U https://publications.rwth-aachen.de/record/61252