Abstract
Frizzled 8-associated Antiproliferative Factor (APF) is a sialoglycopeptide urinary biomarker of interstitial cystitis/painful bladder syndrome (IC/PBS), a chronic condition of unknown etiology with variable symptoms that generally include pelvic and/or perineal pain, urinary frequency, and urgency. We previously reported that native human APF suppresses the proliferation of normal bladder epithelial cells through a mechanism that involves increased levels of p53. The goal of this study was to delineate the regulatory mechanism whereby p53 expression is regulated by APF. Two APF-responsive cell lines (T24 bladder carcinoma cells and the immortalized human bladder epithelial cell line, TRT-HU1) were treated with asialo-APF (as-APF), a chemically synthesized form of APF. Biochemical analysis revealed that as-APF increased p53 levels in two ways: by decreasing ubiquitin specific protease 2a (USP2a) expression leading to enhanced ubiquitination of murine double minute 2 E3 ubiquitin ligase (MDM2), and by suppressing association of p53 with MDM2, thus impairing p53 ubiquitination. Biological responses to as-APF were suppressed by increased expression of wild type, but not mutant USP2a, which enhanced cell growth via upregulation of a cell cycle mediator, cyclin D1, at both transcription and protein levels. Consistent with this, gene silencing of USP2a with siRNA arrested cell proliferation. Our findings suggest that APF upregulates cellular p53 levels via functional attenuation of the USP2a-MDM2 pathway, resulting in p53 accumulation and growth arrest. These data also imply that targeting USP2a, MDM2, p53 and/or complex formation by these molecules may be relevant in the development of novel therapeutic approaches to IC/PBS.
Highlights
More than one out of 77 people (3–8 million women and 1–4 million men) have been diagnosed with interstitial cystitis/painful bladder syndrome (IC/PBS) in the U.S [1,2], resulting in a great public health burden [3,4,5]
Anti-proliferative factor (APF) has been directly implicated in the pathogenesis of IC/PBS, as bladder epithelial cells from IC/PBS patients that produce this factor consistently proliferate at an abnormally slow rate compared to explanted epithelial cells from matched controls, and because purified human APF is a bladder epithelial cell growth inhibitor [15,19]
Protein extracts were prepared from growing TRT-HU1, RT4 and T24 bladder cell lines for western blot analysis using antibodies against murine double minute 2 E3 ubiquitin ligase (MDM2), p53 and b-actin. (B) MDM2, an E3 ubiquitin ligase, is decreased in response to as-APF
Summary
More than one out of 77 people (3–8 million women and 1–4 million men) have been diagnosed with interstitial cystitis/painful bladder syndrome (IC/PBS) in the U.S [1,2], resulting in a great public health burden [3,4,5]. Anti-proliferative factor (APF), a short sialoglycopeptide whose primary structure is 100% homologous to the putative sixth transmembrane domain of frizzled 8 (a Wnt receptor), has been proposed as a candidate biomarker for IC/PBS, based on the finding of significantly increased APF activity levels in urine from IC/PBS patients compared to normal controls [17,18]. APF has been directly implicated in the pathogenesis of IC/PBS, as bladder epithelial cells from IC/PBS patients that produce this factor consistently proliferate at an abnormally slow rate compared to explanted epithelial cells from matched controls, and because purified human APF is a bladder epithelial cell growth inhibitor [15,19]. Decreased cell proliferation along with reduced tight junction protein expression in response to APF is consistent with thinning, denudation and increased permeability observed in bladder epithelium of IC/PBS patients [20,21]
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