No 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 ␤-catenin was a prominent node in this network. Functional assays demonstrated that APF down-regulated ␤-catenin, at least in part, via proteasomal and lysosomal degradation. Moreover, silencing of ␤-catenin mimicked the antiproliferative effect of APF whereas ectopic expression of nondegradable ␤-catenin rescued growth inhibition in response to APF, confirming that ␤-catenin is a key mediator of APF signaling. Notably, the key role of ␤-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 ␤-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 ␤-catenin elevated COX-2 expression, whereas forced expression of nondegradable ␤-catenin inhibited APF-induced up-regulation of COX-2. Furthermore, we confirmed that ␤-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 ␤-catenin is a key node, and provides new insight that targeting the ␤-catenin signaling pathway may be a rational approach toward treating interstitial cystitis. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.007492, 1-11, 2011. Antiproliferative factor (APF)1 , a nine-residue sialoglycopeptide whose peptide chain is 100% homologous to the putative sixth transmembrane domain of , is secreted by bladder epithelial cells from patients with interstitial cystitis (IC) (2, 3), a prevalent and debilitating pelvic disorder (4, 5). Studies suggest that APF is a potent negative growth factor, which markedly inhibits the proliferation of not only normal bladder epithelial cells but also T24 bladder carcinoma cells and HeLa cervical carcinoma cells (1, 6, 7). Studies have been conducted to investigate the molecular mechanisms underlying the antiproliferative effect of APF using the hypothesis-driven approach; these led to the discoveries that (a) cytoskeleton-associated protein 4 (CKAP4), also known as CLIMP63, is a high-affinity receptor for APF (6); (b) palmitoylation of CKAP4 by ...

Transfusion of red blood cells (RBCs) is a common life-saving clinical practice in severely anemic or hemorrhagic patients; however, it may result in serious pathological complications such as transfusion-related acute lung injury. The factors mediating the deleterious effects of RBC transfusion remain unclear. In this study, we tested the effects of washed long-term (RBC-O; >28 days) versus short-term (RBC-F; <14 days) stored RBCs and their supernatants on lung endothelial (EC) permeability under control and inflammatory conditions. RBCs enhanced basal EC barrier function as evidenced by an increase in transendothelial electrical resistance and decrease in permeability for macromolecules. RBCs also attenuated EC hyperpermeability and suppressed secretion of EC adhesion molecule ICAM-1 and proinflammatory cytokine IL-8 in response to LPS or TNF-α. In both settings, RBC-F had slightly higher barrier protective effects as compared with RBC-O. In contrast, supernatants from both RBC-F and RBC-O disrupted the EC barrier. The early phase of EC permeability response caused by RBC supernatants was partially suppressed by antioxidant N-acetyl cysteine and inhibitor of Src kinase family PP2, while addition of heme blocker and inhibition of NOD-like receptor family pyrin domain containing protein 3 (NLRP3), stress MAP kinases, receptor for advanced glycation end-products (RAGE), or Toll-like receptor-4 (TLR4) signaling were without effect. Morphological analysis revealed that RBC supernatants increased LPS- and TNF-α-induced breakdown of intercellular junctions and formation of paracellular gaps. RBC supernatants augmented LPS- and TNF-α-induced EC inflammation reflected by increased production of IL-6, IL-8, and soluble ICAM-1. These findings demonstrate the deleterious effects of RBC supernatants on EC function, which may have a major impact in pathological consequences associated with RBC transfusion.

Proinflammatory cytokines such as IL-6 induce endothelial cell (EC) barrier disruption and trigger an inflammatory response in part by activating the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. The protein suppressor of cytokine signaling-3 (SOCS3) is a negative regulator of JAK-STAT, but its role in modulation of lung EC barrier dysfunction caused by bacterial pathogens has not been investigated. Using human lung ECs and EC-specific SOCS3 knockout mice, we tested the hypothesis that SOCS3 confers microtubule (MT)-mediated protection against endothelial dysfunction. SOCS3 knockdown in cultured ECs or EC-specific SOCS3 knockout in mice resulted in exacerbated lung injury characterized by increased permeability and inflammation in response to IL-6 or heat-killed Staphylococcus aureus (HKSA). Ectopic expression of SOCS3 attenuated HKSA-induced EC dysfunction, and this effect required assembled MTs. SOCS3 was enriched in the MT fractions, and treatment with HKSA disrupted SOCS3–MT association. We discovered that—in addition to its known partners gp130 and JAK2—SOCS3 interacts with MT plus-end binding proteins CLIP-170 and CLASP2 via its N-terminal domain. The resulting SOCS3–CLIP-170/CLASP2 complex was essential for maximal SOCS3 anti-inflammatory effects. Both IL-6 and HKSA promoted MT disassembly and disrupted SOCS3 interaction with CLIP-170 and CLASP2. Moreover, knockdown of CLIP-170 or CLASP2 impaired SOCS3–JAK2 interaction and abolished the anti-inflammatory effects of SOCS3. Together, these findings demonstrate for the first time an interaction between SOCS3 and CLIP-170/CLASP2 and reveal that this interaction is essential to the protective effects of SOCS3 in lung endothelium.

Suppressors of cytokine signaling (SOCS) provide negative regulation of inflammatory reaction. The role and precise cellular mechanisms of SOCS1 in control of endothelial dysfunction and barrier compromise associated with acute lung injury remain unexplored. Our results show that siRNA‐mediated SOCS1 knockdown augmented lipopolysaccharide (LPS)‐induced pulmonary endothelial cell (EC) permeability and enhanced inflammatory response. Consistent with in vitro data, EC‐specific SOCS1 knockout mice developed more severe lung vascular leak and accumulation of inflammatory cells in bronchoalveolar lavage fluid. SOCS1 overexpression exhibited protective effects against LPS‐induced endothelial permeability and inflammation, which were dependent on microtubule (MT) integrity. Biochemical and image analysis of unstimulated EC showed SOCS1 association with the MT, while challenge with LPS or MT depolymerizing agent colchicine impaired this association. SOCS1 directly interacted with N2 domains of MT‐associated proteins CLIP‐170 and CLASP2. Furthermore, N‐terminal region of SOCS1 was indispensable for these interactions and SOCS1‐ΔN mutant lacking N‐terminal 59 amino acids failed to rescue LPS‐induced endothelial dysfunction. Depletion of endogenous CLIP‐170 or CLASP2 abolished SOCS1 interaction with Toll‐like receptor‐4 and Janus kinase‐2 leading to impairment of SOCS1 inhibitory effects on LPS‐induced inflammation. Altogether, these findings suggest that endothelial barrier protective and anti‐inflammatory effects of SOCS1 are critically dependent on its targeting to the MT.