Browsing Theses and Dissertations School of Pharmacy by Subject "retinoic acid"
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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.
Targeting the Activator Protein-1 Complex to Inhibit Airway Smooth Muscle Cell Hyperproliferation in AsthmaHyperproliferation of airway smooth muscle (ASM) cells leads to increased ASM mass causing airway obstruction in inflammatory diseases such as asthma. Currently, there are no effective therapies to modulate ASM cell proliferation that contributes to debilitating bronchoconstriction in severe asthmatics. Previous studies suggest that activator protein-1 (AP-1) transcription factor expression is upregulated in airway cells in asthma and inhibition of AP-1 could mitigate the hyperproliferation of ASM cells. AP-1 activity has been shown to be enhanced by upstream extracellular signal-regulated kinase (ERK1/2) signaling or antagonized by retinoic acid receptor (RAR)-mediated signaling. The overall goal of the current study was to evaluate the therapeutic potential of a combination therapy of an ERK1/2 inhibitor and RAR agonist to modulate AP-1 complex formation and activation. Aim 1 studies tested the hypothesis that a novel function-selective ERK1/2 inhibitor, referred to as SF-3-030, would mitigate off-target toxicity while regulating platelet-derived growth factor (PDGF) induced AP-1 activity and ASM cell proliferation. In Aim 2 studies we evaluated the role of retinoids in controlling AP-1 complex formation and identified a RARγ isoform-specific agonist, CD1530, as a potential therapeutic option for inhibition of AP-1 activity and ASM cell hyperproliferation. Aim 3 studies determined whether a polypharmacological approach of combining ERK1/2 inhibition and RAR agonism to target two different aspects of the AP-1 complex activation and formation would have an additive effect in preventing ASM hyperproliferation. Overall, these studies help further our understanding of how AP-1 signaling causes the hyperproliferation of ASM cells while elucidating possible therapeutic treatment options through ERK1/2 inhibition and RAR agonism.