title: Antiproliferative factor regulates connective tissue growth factor (CTGF/CCN2) expression in T24 bladder carcinoma cells.
abstract: Antiproliferative factor (APF) is a sialoglycopeptide elevated in the urine of patients with interstitial cystitis (IC)-a chronic, painful bladder disease of unknown etiology. APF inhibits the proliferation of normal bladder epithelial and T24 bladder carcinoma cells in vitro by binding to cytoskeleton-associated protein 4 (CKAP4) and altering the transcription of genes involved in proliferation, cellular adhesion, and tumorigenesis; however, specific molecular mechanisms and effector genes that control APF's antiproliferative effects are unknown. In this study, we found that there was a 7.5-fold up-regulation of connective tissue growth factor (CTGF/CCN2) expression in T24 bladder carcinoma cells treated with APF. Western blot revealed a dose-dependent increase in CCN2 protein levels, with secretion into the culture medium after APF treatment. CCN2 overexpression enhanced APF's antiproliferative activity, whereas CCN2 knockdown diminished APF-induced p53 expression. Using a luciferase reporter construct, we found that APF treatment resulted in fivefold activation of the CCN2 proximal promoter and, of importance, that small interfering RNA-mediated knockdown of CKAP4 inhibited CCN2 upregulation. In addition, we demonstrate that CKAP4 translocates to the nucleus and binds to the CCN2 proximal promoter in an APF-dependent manner, providing evidence that CCN2 regulation by APF involves CKAP4 nuclear translocation and binding to the CCN2 promoter.
authors.affiliation: Department of Basic Sciences, The Commonwealth Medical College, Scranton, PA 18509, USA.
authors.forename: Christina A
authors.initials: CA
authors.lastname: Matika
authors.forename: Melissa
authors.initials: M
authors.lastname: Wasilewski
authors.forename: John A
authors.initials: JA
authors.lastname: Arnott
authors.forename: Sonia Lobo
authors.initials: SL
authors.lastname: Planey
mesh_terms: D021581:Active Transport, Cell Nucleus; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D055513:Connective Tissue Growth Factor / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D034622:RNA Interference; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
chemical_list: C526317:CCN2 protein, human; C081876:CKAP4 protein, human; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C490708:antiproliferative factor APF, human; D055513:Connective Tissue Growth Factor