Date of Award

5-1-2014

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Cao, Deliang

Abstract

Aldo-keto reductase 1B10 (AKR1B10) promotes cell growth and proliferation via eliminating cytotoxic carbonyls and mediating fatty acid/lipid synthesis, and induces drug resistance to cytostatic antitumor agents containing carbonyl groups such as daunorubicin, resulting in failure of treatment. AKR1B10 may also participate in cell differentiation and carcinogenesis by depleting the precursor of cellular retinoic acid, a signaling molecule, and activating pro-carcinogens, such as polycyclic aromatic hydrocarbon (PAH). AKR1B10 is primarily expressed in human colonic mucosa, but its biochemistry, biological functions, and pathological roles in normal and diseased conditions of the colon remain unclear. In this study, we characterized the biochemical properties of AKR1B10 protein and investigated its pathophysiological roles in colonic diseases. The reversible thiol/disulfide exchange is an important regulatory mechanism of protein enzymatic activity. Many protein enzymes are susceptible to S-thiolation induced by reactive oxygen species (ROS); and physiological thiols, glutathione (GSH) and free amino acid cysteine (Cys), are critical intracellular thiol antioxidants, protecting proteins from irreversible oxidative damage. AKR1B10 contains 4 Cys residues, i.e., Cys45, Cys187, Cys200, and Cys299; and in the 3D structure, the Cys299, a key residue for AKR1B10 enzymatic activity, is located to the Cys187 at 10.45 Å. Exposing AKR1B10 to ROS resulted in significant decrease of its free sulfhydryl groups, up to 40% and 50% when 0.5 or 1.0 mM Cys was used in the ROS mixture, respectively; accordingly, AKR1B10 activity was decreased in parallel with the oxidation of the sulfhydryl groups, ranked as ROS with Cys >> ROS with GSH > ROS. ROS-induced thiolation also affected the sensitivity of AKR1B10 to inhibitors EBPC, epalrestat, and statil. Ulcerative colitis (UC) and colitis-associated colorectal cancer (CAC) is a serious health problem, but etiopathological factors remain unclear. AKR1B10 was lost or remarkably decreased in more than 90% of UC and CAC tissue, but the causative role of AKR1B10 in colitis and its associated cancer is unknown. Aldo-keto reductase 1B8 (AKR1B8) is an orthologue of human AKR1B10 in the mouse. This study produced a new AKR1B8 knockout (-/-) mouse strain to investigate the pathogenic role of AKR1B10 in human colitis and associated cancer. We found that targeted disruption of AKR1B8 led to decrease in lipid synthesis from butyrate and defects in cryptic development and aberrations of cryptic cell proliferation, migration, and maturation, disrupting dynamic homeostasis of self-renewal. AKR1B8 deficient mice were highly susceptible to colitis induced by dextran sulfate sodium at a lower dose (2%) and demonstrated severe liquid diarrhea and gross bleeding. Histopathologically, AKR1B8 -/- mucosa had drastic inflammatory cell infiltration, ulceration and fiber deposits; injury repair of epithelium was impaired in AKR1B8 -/- mucosa, and ulcers are healed by scarring, forming rigid colon tubes. Colitis-associated tumorigenesis occurred frequently and multiplically in AKR1B8 -/- mucosa, and palpable masses appeared in AKR1B8 -/- mice at the early stage of colitis, but not in wild type control mice. Exome sequencing revealed that AKR1B8 -/- genome had severe carbonyl and oxidative-induced DNA damage. These data suggest that AKR1B8 is a critical protein that modulates the self-renewal of crypt cells and susceptibility of colon mucosa to colitis and associated tumorigenesis. In summary, our data revealed for the first time that AKR1B10 enzymatic activity and inhibitor sensitivity are reversibly modulated by thiol/disulfide exchanges. More importantly, AKR1B8 is a critical protein that regulates the homeostasis and entirety of colonic epithelium. AKR1B8 deficiency in epithelium is a critical etiopathogenic factor of UC and CAC.

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