Antiproliferative factor regulates connective tissue growth factor (CTGF/CCN2) expression in T24 bladder carcinoma cells

Abstract

Connective tissue growth factor (CTGF/CNN2) is a novel APF target gene. A novel mechanism is described by which the APF cellular receptor, cytoskeleton-associated protein 4 (CKAP4), mediates APF-induced CTGF transcription.

Cytoskeleton-Associated Protein 4: Functions Beyond the Endoplasmic Reticulum in Physiology and Disease

“…Evidence confirming CKAP4's ability to function as a receptor for APF was exhibited by the use of an antibody against CKAP4 which blocked APF activity and by siRNA knockdown of CKAP4 which reduced cell sensitivity to APF. Subsequent studies confirmed the cell surface expression of CKAP4 in HeLa cells by immunolocalization of nonpermeabilized cells [14] and by cell surface biotinylation [16] and also demonstrated that CKAP4 expression was required for APF-induced cellular effects, including (1) inhibition of normal bladder epithelial cell and cervical and bladder carcinoma cell proliferation [11,[13][14][15], (2) altered gene expression (i.e., vimentin, zonular occludens-1 [ZO-1], E-cadherin, p53, MMP2, and CCN2 [13][14][15]), and (3) Akt/GSK3β/β-catenin phosphorylation [15].…”

“…In direct support of the idea that CKAP4 must be phosphorylated to enter the nucleus, Zacharias et al also showed by metabolic labeling with γ 32 P-ATP that nuclear CKAP4 is phosphorylated [16]. Matika et al (2012) reported similar nuclear localization of CKAP4 in response to APF in T24 bladder carcinoma cells using confocal microscopy and cellular fractionation followed by Western Blot [13]. Of significant importance in this study was the detection of a second, smaller CKAP4-specific band in the nuclear fraction of APF-treated cells, suggesting that CKAP4 may be proteolyzed prior to entering the nucleus; moreover, the detection of a relatively small amount of CKAP4 in the nucleus of untreated cells in this study and in other unpublished experiments suggests that CKAP4 may have some inherent nuclear function not related to APF-induced signal transduction or that may be specific to cancer cells, as nuclear localization of CKAP4 after binding of tPA or SPA was not apparent in previous studies [10,12].…”

“…Interestingly, there is new evidence demonstrating CKAP4 localization in the nucleus of both HeLa [14,16] and T24 bladder [13] Importantly, this APF-induced change in CKAP4 localization was shown to be dependent on palmitoylation of CKAP4 by DHHC2 [14] (Figure 2). To further test the idea that CKAP4 translocates to the nucleus in response to APF, Zacharias et al (2012) used three different techniques-surface labeling with Sulfo-NHS-biotin, cellular fractionation, and immunocytochemistry followed by confocal microscopy-to monitor CKAP4 localization in response to APF binding [16].…”

“…(e) The SP-A/CKAP4 interaction is recycled via a clathrin pathway and is stimulated by the cAMP signaling cascade [18]. (f) CKAP4 binds to the CCN2 promoter via its C-terminal domain (residues 126-501 which includes the bZIP-like DNA-binding domain) in an APF-dependent fashion [13]. It is not known whether APF binds CKAP4 in the nucleus.…”

“…For example, CKAP4 has been shown to be required for APF-mediated signaling [11,[13][14][15][16], for gentamycin-induced cytotoxicity [17], for regulation of tPA function [10], and for SP-A-induced surfactant uptake by pneumocytes [18]. This evidence for CKAP4 mediating a wide array of cellular responses important in physiological and pathological processes, such as interstitial cystitis, druginduced cytotoxicity, pericullar proteolytic activity, and lung lipid homeostasis, provides new insights into this classically ER-resident protein.…”