Abstract
Antiproliferative factor (APF) is a low molecular weight sialoglycopeptide that is secreted by bladder cells from interstitial cystitis patients and is a potent inhibitor of both normal bladder epithelial and bladder carcinoma cell proliferation. We hypothesized that APF may produce its antiproliferative effects by binding to a transmembrane receptor. This study demonstrates that cytoskeleton-associated protein 4/p63 (CKAP4/p63), a type II transmembrane receptor, binds with high affinity to APF. The antiproliferative activity of APF is effectively inhibited by preincubation with anti-CKAP4/p63-specific antibodies, as well as by short interfering RNA knockdown of CKAP4/p63. Immunofluorescent confocal microscopy showed co-localization of anti-CKAP4/p63 and rhodamine-labeled synthetic APF binding in both cell membrane and perinuclear areas. APF also inhibits the proliferation of HeLa cervical carcinoma cells that are known to express CKAP4/p63. These data indicate that CKAP4/p63 is an important epithelial cell receptor for APF.
Highlights
Antiproliferative factor (APF)2 is a sialoglycopeptide inhibitor of bladder epithelial cell proliferation that is secreted by bladder epithelial cells from patients with interstitial cystitis (IC) [1, 2], a disorder commonly associated with denudation or thinning of the bladder epithelium [3,4,5]
Because decreased urine levels of heparin-binding epidermal growth factor-like growth factor [10], bladder epithelial thinning or ulceration [3, 4], abnormal expression of some of the same proteins [11], and bladder epithelial leakiness [12] have all been described in IC patients in vivo, APF appears to play a pivotal role in the pathogenesis of IC
This study presents evidence that CKAP4/p63 is a functional bladder epithelial cell receptor for APF, an inhibitor of cell proliferation secreted from bladder epithelial cells in patients suffering from the chronic painful bladder disorder called interstitial cystitis
Summary
Cell Cultures—Explanted bladder epithelial cells were propagated from biopsies of four patients who had undergone cystoscopy and fulfilled the NIDDK/National Institutes of Health diagnostic criteria for interstitial cystitis [13] and their age-, race-, and gender-matched controls. The microsomal fraction was solubilized in PBS, pH 7.2, containing 1 mM NaVO3, 10 mM NaF, 1 mM EDTA, 0.1 mM PMSF, and 1% Triton X-100 This solution was allowed to incubate on ice for 30 min and diluted to 0.5% Triton X-100 with ice-cold wash buffer. Nanoflow Reverse Phase Liquid Chromatography-Tandem Mass Spectrometry—Chromatographic separations of the tryptic peptides were conducted using a microcapillary column with an integrated electrospray ionization (ESI) emitter constructed by flame-pulling a fine tip (ϳ5–7-mm orifice) on a 75-mm inner diameter ϫ 360-mm outer diameter ϫ 10-cm long segment of fused silica (Polymicro Technologies Inc., Phoenix, AZ). Primary normal bladder epithelial cells were trypsinized for 10 min at room temperature and centrifuged in growth medium
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