Login to Access Video or Poster Abstract: MP82-04
Sources of Funding: Research reported in this publication was supported by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Number F31AI106357.

Introduction

Interstitial cystitis (IC) patients suffer from chronic pelvic pain and bladder dysfunction. IC patients have altered cortisol levels suggesting dysregulation of the hypothalamic-pituitary adrenal (HPA) axis and suffer from exacerbated symptoms in response to high stress. Corticotropin-releasing factor (CRF) is the initiator of the HPA axis and mediates stress responses and voiding control, where increased CRF levels in Barrington's nucleus induce bladder dysfunction. Arachidonic acid (AA) metabolites have been shown to induce CRF expression, however the transcriptional mediators of this modulation are unknown. Here we identify transcription factors that mediate AA-induced CRF gene expression.

Methods

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 to evaluate responses to AA and the impact of AhR and PPAR gamma expressed in HEK 293T cells. The hypothalamic neuronal cell line N42 was used to evaluate the role of AhR and PPAR gamma in native promoter regulation by RT PCR. We also generated mice that have PPAR gamma and AhR knocked-out in CRF-expressing cells and characterized voiding activity._x000D_ _x000D_

Results

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 PPAR gamma alone showed no change, while over-expression of AhR alone showed a significant increase in AA-induced CRF expression; this was inhibited by over-expression of both AhR and PPAR gamma in HEK 293T cells. Over-expression of AhR in N42 cells resulted in increased CRF mRNA in response to AA induction. AhR conditional knockout mice showed increased voiding frequency.

Conclusions

These results suggest AhR binding to the XRE sites modulates AA-dependent CRF gene expression. PPAR gamma inhibits AA-dependent CRF gene expression. Continued studies will use mouse models to examine the in vivo role of AhR and PPAR gamma inhibitors to modulate voiding activity.

Funding

Research reported in this publication was supported by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Number F31AI106357.