Abstract
Antiproliferative factor (APF), a Frizzled-8 protein-related sialoglycopeptide involved in the pathogenesis of interstitial cystitis, potently inhibits proliferation of normal urothelial cells as well as certain cancer cells. To elucidate the molecular mechanisms of the growth-inhibitory effect of APF, we performed stable isotope labeling by amino acids in cell culture analysis of T24 bladder cancer cells treated with and without APF. Among over 2000 proteins identified, 54 were significantly up-regulated and 48 were down-regulated by APF treatment. Bioinformatic analysis revealed that a protein network involved in cell adhesion was substantially altered by APF and that β-catenin was a prominent node in this network. Functional assays demonstrated that APF down-regulated β-catenin, at least in part, via proteasomal and lysosomal degradation. Moreover, silencing of β-catenin mimicked the antiproliferative effect of APF whereas ectopic expression of nondegradable β-catenin rescued growth inhibition in response to APF, confirming that β-catenin is a key mediator of APF signaling. Notably, the key role of β-catenin in APF signaling is not restricted to T24 cells, but was also observed in an hTERT-immortalized human bladder epithelial cell line, TRT-HU1. In addition, the network model suggested that β-catenin is linked to cyclooxygenase-2 (COX-2), implying a potential connection between APF and inflammation. Functional assays verified that APF increased the production of prostaglandin E(2) and that down-modulation of β-catenin elevated COX-2 expression, whereas forced expression of nondegradable β-catenin inhibited APF-induced up-regulation of COX-2. Furthermore, we confirmed that β-catenin was down-regulated whereas COX-2 was up-regulated in epithelial cells explanted from IC bladder biopsies compared with control tissues. In summary, our quantitative proteomics study describes the first provisional APF-regulated protein network, within which β-catenin is a key node, and provides new insight that targeting the β-catenin signaling pathway may be a rational approach toward treating interstitial cystitis.
Highlights
From the ‡The Urological Diseases Research Center, Children’s Hospital Boston, Boston, MA 02115, USA, §Departments of Surgery and Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA, ¶Proteomics Center, Children’s Hospital Boston, Boston, MA 02115, USA, ʈDivision of Molecular Life Sciences, Pohang University of Science and Technology, Pohang, Kyungbuk, 790-784, Republic of Korea, **Division of Infectious Diseases, Department of Medicine, the University of Maryland School of Medicine and VA Medical Center, Baltimore, MD 21201, USA, ‡‡Department of Pathology, the University of Maryland School of Medicine, Baltimore, MD 21201, USA, §§Department of Molecular Biotechnology, WCU Program, Institute of Biomedical Science and Technology, Konkuk University, Seoul, Republic of Korea, ¶¶Department of Pathology, Children’s Hospital Boston and Harvard Medical School, Boston, MA, 02115, USA
DHHC2 plays a critical role in regulating Antiproliferative factor (APF)-mediated signaling [7]; (c) APF inhibits the production of the urothelial cell mitogen, heparin-binding epidermal growth factor-like growth factor (HB-EGF) [8]; (d) HB-EGF functionally antagonizes APF activity [8] via parallel mitogen-activated protein kinase signaling pathways [9]; and (e) the transcription factor p53 is an important mediator of APF-induced growth inhibition [10]
It is generally believed that SILAC labeling will not alter protein levels, quantitative analyses of proteins extracted from control cells cultured in parallel in “light” and “heavy” media have shown that levels of a minor fraction of proteins can be significantly changed by differential labeling [24, 27]
Summary
Reagents—APF peptides were purified from the supernatant of bladder epithelial cells explanted from IC patients using molecular weight fractionation, ion exchange chromatography, hydrophobic interaction chromatography, and reversed-phase high-performance liquid chromatography [1]. For SILAC labeling, 1 ϫ 105 T24 cells were grown in arginine- and lysine-depleted medium supplemented with 10% (v/v) dialyzed FBS, and either Arg0 and Lys0 or Arg and Lys. For SILAC labeling, 1 ϫ 105 T24 cells were grown in arginine- and lysine-depleted medium supplemented with 10% (v/v) dialyzed FBS, and either Arg0 and Lys0 or Arg and Lys8 Both “light” and “heavy” culture media were replaced every 2 days. Transient Cell Transfection—T24 bladder cells were grown to ϳ80% confluence in 100-mm dishes, at which time they were transiently transfected with small interfering RNAs (siRNAs) against -catenin or OFF-TARGET controls using Lipofectamine 2000 according to the manufacturer’s instructions. Data were compared using a paired Student’s t test. p Ͻ 0.05 was considered to be statistically significant
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