Browsing Theses and Dissertations School of Pharmacy by Title "Impact of cellular retinol-binding protein, type I on retinoic acid biosynthesis and homeostasis"
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Impact of cellular retinol-binding protein, type I on retinoic acid biosynthesis and homeostasisStatement: A global Rbp1 knock out (Rbp1-/-) mouse model was used to correlate direct retinoid measurements with vitamin A metabolizing and atRA biosynthesizing enzyme activities, Crbp function and tissue microenvironment for the first time. Methods: atRA was quantified by LC-MRM3 and ROL/RE/RAL was quantified by HPLC-UV. Enzyme activities were measured from enzymes present in subcellular fractions isolated from WT and Rbp1-/- tissues. Mouse CrbpI and CrbpIII were purified from transformed Escherichia coli for functional comparative studies. Tissue were formalin fixed for histological examination. Relative gene expression was analyzed using quantitative PCR. Results: Reduced atRA was consistently quantified in extrahepatic tissues with elevated ROL/RE. Relative gene expression showed altered expression in retinoid pathway proteins and atRA loss preceded expression changes in some cases. Tissue microenvironments also consistently showed a loss of structure and organization along with accumulation of extracellular matrix and hyperplasia without apparent disease. Functional studies showed that CrbpIII binds retinol with less affinity than CrbpI and does not function equivalently to CrbpI in regulation of atRA biosynthesis. Also, metabolizing enzymes had altered activities in the Rbp1-/- tissues with reduced atRA biosynthesis. Conclusions: Loss of CrbpI results in altered regulation of enzyme activity and atRA homeostasis cannot be maintained by other Crbp homologs in extrahepatic tissues. Dysfunctional atRA biosynthesis due to loss of CrbpI results in altered tissue microenvironment characteristic of dietary vitamin A deficiency and precancerous dysfunction associated with cancers that are observed to have silenced CrbpI.