AOAH AS A MODULATOR OF CORTICOTROPHIN RELEASING FACTOR

Introduction: Through a genetic screen we have identified acyloxyacyl hydrolase (AOAH) as a genetic determinant of pelvic hypersensititvity. Aoah-/- mice suffer from chronic pelvic pain, bladder dysfunction, and have increased arachidonic acid (AA) levels in their spinal cord. Corticotropin-releasing factor (CRF) has been well established as a key mediator of stress responses and voiding control, where increased CRF levels in Barrington’s nucleus induce bladder dysfunction. AA and AA metabolites have been shown to modulate CRF expression, however the transcriptional mediators of this modulation are unknown. We are investigating the role of AOAH in modulating CRF and identifying which transcription factors mediate AA-induced CRF gene expression. Methods: We tested female Aoah-/- mice relative to wild type (WT) for voiding activity through awake cystometry, and by the blotting paper method. We evaluated depression-like behavior using a novelty suppressed feeding (NSF) assay, and sucrose preference test. We quantified CRF mRNA expression through RT PCR of 1mM brain sections from the paraventricular nucleus and Barrington’s nucleus in WT and Aoah-/- mice. We also over expressed the human AOAH expression construct and tested for AA-dependent CRF-promoter activity. To identify the transcription factors that mediate AA-dependent CRF expression we used MIRAGE software to identify candidate transcription factor binding sites in a 1kb region of the human CRF gene promoter. We identified a peroxisome proliferator−activated hormone response element (PPRE) and two Xenobiotic Responsive Element (XRE) sites as candidate mediators of AA−dependent CRF induction. Site−directed mutations of the PPRE and XRE sites were generated in a CRF−luciferase reporter plasmid. We evaluated expression of WT CRF and the mutants in HEK 293T cells for their responses to AA. Results: Aoah-/- mice void significantly fewer times than WT mice with significantly larger volumes and showed more non-voiding bladder contractions. In the NSF assay Aoah-/- mice take significantly longer to approach the food pellet and to consume the food pellet; Aoah-/- mice consumed significantly less sucrose water compared to WT mice consistent with increased depression-like behavior. Aoah-/- mice have significantly increased CRF mRNA levels in the paraventricular nucleus and Barrington’s nucleus. Co-transfection of AOAH resulted in a significant increase in AA-dependent CRF expression. AA induction in the PPRE mutant resulted in increased CRF promoter activity compared to WT, whereas XRE1 had decreased activity. The mutation of both XRE1 and XRE2 resulted in decreased responsiveness to AA. Over-expression of AhR and AOAH showed a significant increase in AA−induced CRF expression. Conclusions: These results suggest AOAH modulates AA-dependent CRF gene expression in an AhR dependent manner. Thus the AOAH/CRF axis is a novel therapeutic target to treat voiding activity and depression.