177

AGER

Fibrosis of the lung is characterized by the accumulation of myofibroblasts, a key mediator in the fibrogenic reaction. Cumulative evidence indicates that epithelial-mesenchymal transition (EMT), a process whereby epithelial cells become mesenchyme-like, is an important contributing source for the myofibroblast population. Underlying this phenotypical change is a dramatic alteration in cellular structure. The receptor for advanced glycation end-products (RAGE) has been suggested to maintain lung homeostasis by mediating cell adhesion, while the family of ezrin/radixin/moesin (ERM) proteins, on the other hand, serve as an important cross-linker between the plasma membrane and cytoskeleton. In the present investigation, we tested the hypothesis that RAGE and ERM interact and play a key role in regulating EMT-associated structural changes in alveolar epithelial cells. Exposure of A549 cells to inflammatory cytokines resulted in phosphorylation and redistribution of ERM to the cell periphery and localization with EMT-related actin stress fibers. Simultaneously, blockade of Rho kinase (ROCK) signaling attenuated these cytokine-induced structural changes. Additionally, RAGE expression was diminished after cytokine stimulation, with release of its soluble isoform via a matrix metalloproteinase (MMP)-9-dependent mechanism. Immunofluorescence microscopy and coimmunoprecipitation revealed association between ERM and RAGE under basal conditions, which was disrupted when challenged with inflammatory cytokines, as ERM in its activated state complexed with membrane-linked CD44. Dual-fluorescence immunohistochemistry of patient idiopathic pulmonary fibrosis (IPF) tissues highlighted marked diminution of RAGE in fibrotic samples, together with enhanced levels of CD44 and double-positive cells for CD44 and phospho (p)ERM. These data suggest that dysregulation of the ERM-RAGE complex might be an important step in rearrangement of the actin cytoskeleton during proinflammatory cytokine-induced EMT of human alveolar epithelial cells.

S100A8/A9 proteins are members of EF-hand calcium-binding proteins secreted by neutrophils and activated monocytes. S100A8/A9 has cell growth-promoting activity at low concentrations by binding to the receptor for advanced glycation end products (RAGE). In this study, we report for the first time that S100A8/A9 promoted the invasion of breast cancer cells depending on RAGE. In addition, RAGE binding to S100A8/A9 promoted the phosphorylation of LIN-11, Isl1, and MEC-3 protein domain kinase, as well as cofilin. This phosphorylation is a critical step in cofilin recycling and actin polymerization. Interestingly, RAGE binding to S100A8/A9 enhanced cell mesenchymal properties and induced epithelial-mesenchymal transition. Mechanistically, RAGE binding to S100A8/A9 stabilized Snail through the NF-kappaB signaling pathway. Based on these observations, RAGE expression in breast cancer cells was associated with lymph node and distant metastases in patients with invasive ductal carcinoma. Moreover, RAGE binding to S100A8/A9 promoted lung metastasis in vivo. In summary, our in vitro and in vivo results indicated that RAGE binding to S100A8/A9 played an important role in breast cancer invasion/metastasis. This study identified both RAGE and S100A8/A9 as potential anti-invasion targets for therapeutic intervention in breast cancer.

Basement membrane (BM) proteins accumulate chemical modifications with age. One such modification is glycation, which results in the formation of advanced glycation endproducts (AGEs). In a previous study, we reported that AGEs in the human lens capsule (BM) promote the TGFbeta2-mediated epithelial-to-mesenchymal transition (EMT) of lens epithelial cells, which we proposed as a mechanism for posterior capsule opacification (PCO) or secondary cataract formation. In this study, we investigated the role of a receptor for AGEs (RAGE) in the TGFbeta2-mediated EMT in a human lens epithelial cell line (FHL124). RAGE was present in FHL124 cells, and its levels were unaltered in cells cultured on either native or AGE-modified BM or upon treatment with TGFbeta2. RAGE overexpression significantly enhanced the TGFbeta2-mediated EMT responses in cells cultured on AGE-modified BM compared with the unmodified matrix. In contrast, treatment of cells with a RAGE antibody or EN-RAGE (an endogenous ligand for RAGE) resulted in a significant reduction in the TGFbeta2-mediated EMT response. This was accompanied by a reduction in TGFbeta2-mediated Smad signaling and ROS generation. These results imply that the interaction of matrix AGEs with RAGE plays a role in the TGFbeta2-mediated EMT of lens epithelial cells and suggest that the blockade of RAGE could be a strategy to prevent PCO and other age-associated fibrosis.

Abnormal expression of high-mobility group box-1 (HMGB1) protein occurs in many tumors and is closely associated with tumor invasion and metastasis. However, a role for HMGB1 in epithelial-mesenchymal transition (EMT) in hypopharyngeal carcinoma has not been previously reported. We cultured cells of the hypopharyngeal carcinoma cell line FaDu in vitro and then treated them with 5 ng/ml TGF-beta1 for 48 h to induce EMT. Vimentin, Snail, and HMGB1 expression patterns were then detected using immunofluorescence staining; HMGB1 mRNA and protein expression were verified by RT-PCR and western blot analyses. HMGB1 was then silenced in FaDu cells using RNAi, followed by detection of Vimentin, Snail, and HMGB1 expressions by immunofluorescence staining. The mRNA expression levels of Vimentin, Snail, HMGB1, and E-cadherin were verified by RT-PCR, while protein expression of HMGB1 and receptor for advanced glycation end products (RAGE) were detected by western blot analysis. The biological behavior of FaDu cells was observed before and after HMGB1 silencing using wound healing and cell invasion assays. Following culture with 5 ng/ml TGF-beta1 for 48 h, the morphology of FaDu cells changed from a regular cobblestone-like appearance into a spindle-like shape. expression levels of Vimentin, Snail, and HMGB1 were upregulated at both mRNA and protein levels as determined by RT-PCR, immunofluorescence, and western blotting. After HMGB1 silencing, mRNA expression levels of the epithelial cell marker E-cadherin were upregulated. Meanwhile, expression levels of the mesenchymal markers Vimentin and Snail were decreased. Western blotting revealed that HMGB1 and RAGE were downregulated. RNAi-mediated inhibition of HMGB1 expression decreased the capacities of FaDu cells for invasion and metastasis as determined by wound healing and cell invasion assays. HMGB1 is essential for maintaining the interstitial cell phenotype in TGF-beta1-induced EMT of FaDu cells, and silencing HMGB1 greatly inhibits the invasive and metastatic ability of these cells.

High mobility group box 1 (HMGB1), a critical damage-associated molecular pattern molecule, has been implicated in several inflammatory diseases and cancer types. The overexpression of HMGB1 protein occurs in prostate cancer, and is closely associated with the proliferation and aggressiveness of tumor cells. However, the underlying mechanisms of HMGB1-induced tumor metastasis in prostate cancer remain unclear. In the present study, it was demonstrated that the expression of HMGB1 was high in prostate cancer samples, particularly in the metastatic tissues. Furthermore, recombinant HMGB1 (rHMGB1) enhanced the invasive and metastatic capabilities of the prostate cancer cells. Molecular phenotype alterations of epithelial-to-mesenchymal transition (EMT) and elevated expression levels of matrix metalloproteinase (MMP)-1, -3 and -10 were observed. In addition, advanced glycosylation end-product specific receptor (RAGE) and its downstream molecule nuclear factor (NF)-kappaB pathway were activated during rHMGB1-induced metastasis. Silencing RAGE or NF-kappaB reversed the upregulation of MMP and EMT marker expression levels, thus reducing the migration and invasiveness of tumor cells. Taken together, these results suggest that highly expressed HMGB1 drives EMT and the overexpression of MMP-1, -3, -10 via the RAGE/NF-kappaB signaling pathways, which facilitates the metastasis of prostate cancer and may be a potential therapeutic target for metastatic prostate cancer.

The pathogenesis of pulmonary fibrosis remains unclear. The receptor for advanced glycation end-products (RAGE) is a multi-ligand receptor known to be involved in the process of fibrotic change in several organs, such as peritoneal fibrosis and kidney fibrosis. The aim of this study was to examine the contribution of RAGE during the acute inflammation and chronic fibrotic phases of lung injury induced by intratracheal instillation of bleomycin in mice. Bleomycin-induced lung fibrosis was evaluated in wild-type and RAGE-deficient (RAGE-/-) mice. Bleomycin administration to wild-type mice caused an initial pneumonitis that evolved into fibrosis. While RAGE-/- mice developed a similar early inflammatory response, the mice were largely protected from the late fibrotic effects of bleomycin. The protection afforded by RAGE deficiency was accompanied by reduced pulmonary levels of the potent RAGE-inducible profibrotic cytokines transforming growth factor (TGF)-beta and PDGF. In addition, bleomycin administration induced high mobility group box 1 (HMGB-1) production, one of the ligands of RAGE, from inflammatory cells that accumulated within the air space. Coculture with HMGB-1 induced epithelial-mesenchymal transition (EMT) in alveolar type II epithelial cells from wild-type mice. However, alveolar type II epithelial cells derived from RAGE-/- mice did not respond to HMGB-1 treatment, such that the RAGE/HMGB-1 axis may play an important role in EMT. Also, bleomycin administration induced profibrotic cytokines TGF-beta and PDGF only in wild-type mouse lungs. Our results suggested that RAGE contributes to bleomycin-induced lung fibrosis through EMT and profibrotic cytokine production. Thus, RAGE may be a new therapeutic target for pulmonary fibrosis.