M2-polarized tumor-associated macrophages (TAMs) are key regulators of the link between inflammation and cancer. A negative correlation between infiltration intensity of M2-polarized TAMs and prognosis of pancreatic cancer has been reported. Epithelial-mesenchymal transition (EMT) is an important biological process in the progression of primary tumors toward metastasis. Inflammation-induced EMT has been previously shown, therefore, we hypothesized M2-polarized TAMs could induce EMT in pancreatic cancer. Toll-like receptor 4 (TLR4) signaling has an active role in tumor progression during chronic inflammation and the receptor is primarily expressed on macrophages. Activation of TLR4 on M2-polarized TAMs stimulates an increase in the cytokine interleukin-10 (IL-10); consequently, another aim was to investigate the potential role of TLR4/IL-10 signaling in the EMT of pancreatic cancer. Treatment with IL-4 (20 ng/ml) for 24 h successfully induced the polarization of macrophage cell line RAW 264.7 to M2 phenotype, IL-10 high , IL-12 low , and IL-23 low , and high expression of CD204 and CD206. A coculture system allowed investigation of the roles of M2-polarized TAMs and TLR4/IL-10 signaling in the EMT of Panc-1 and BxPC-3 pancreatic cancer cell lines. Our results showed that coculture with M2-polarized TAMs increased fibroblastic morphology, upregulated mesenchymal markers vimentin and snail at the mRNA and protein levels, and increased proliferation, migration, and metalloproteinase (MMP)2 and MMP9 proteolytic activity in pancreatic cancer cells. Simultaneously, coculture with M2-polarized TAMs decreased the expression of the epithelial marker E-cadherin. Coculture with pancreatic cancer cells increased TLR4 mRNA and protein expression in M2-polarized TAMs. Application of TLR4 siRNA and neutralizing antibodies against TLR4 and IL-10 markedly inhibited E-cadherin reduction and the upregulation of snail and vimentin. Furthermore, activation of TLR4 signaling by lipopolysaccharide profoundly increased the EMT of pancreatic cancer cells. In conclusion, M2-polarized TAMs promoted EMT in pancreatic cancer cells partially through TLR4/IL-10 signaling, suggesting novel therapeutic strategies and enhancing our understanding of M2-polarized TAMs. Pancreatic ductal adenocarcinoma is highly malignant and is resistant to chemo-and radiotherapeutics. 1 Recent evidence suggests the inflammatory environment of the tumor is critical for its progression. 2 Tumor-associated macrophages (TAMs) are derived from circulating monocytes, which are highly abundant within the tumor and provide a key link between inflammation and cancer. 3 TAMs develop a phenotype similar to M2-polarized macrophages and respond to recruitment to the tumor microenvironment. 4 Previous studies have commonly associated elevated numbers of TAMs with tumor progression and poor patient outcome. 5 This association is likely linked to the typical presence of M2-polarized TAMs during cancer invasion, 6 however, the exact mechanisms by which these cells influence the pr...

In parathyroid hormone-related protein 1-84 ] knockin mice, expression of the polycomb protein Bmi-1 is reduced and potentially can mediate the phenotypic alterations observed. We have therefore now examined the skeletal phenotype of Bmi-1 À/À mice in vivo and also assessed the function of bone marrow mesenchymal stem cells (BM-MSCs) from Bmi-1 À/À mice ex vivo in culture.Neonatal Bmi-1 À/À mice exhibited skeletal growth retardation, with reduced chondrocyte proliferation and increased apoptosis.Osteoblast numbers; gene expression of alkaline phosphatase, type I collagen, and osteocalcin; the mineral apposition rate; trabecular bone volume; and bone mineral density all were reduced significantly; however, the number of bone marrow adipocytes and Ppar-g expression were increased. These changes were consistent with the skeletal phenotype observed in the PTHrP(1-84) knockin mouse. The efficiency of colony-forming unit fibroblast (CFU-F) formation in bone marrow cultures was decreased, and the percentage of alkaline phosphatase-positive CFU-F and Runx2 expression were reduced. In contrast, adipocyte formation and Ppar-g expression in cultures were increased, and expression of the polycomb protein sirtuin (Sirt1) was reduced. Reduced proliferation and increased apoptosis of BM-MSCs were associated with upregulation of senescence-associated tumor-suppressor genes, including p16, p19, and p27. Analysis of the skeletal phenotype in Bmi-1 À/À mice suggests that Bmi-1 functions downstream of PTHrP. Furthermore, our studies indicate that Bmi-1 maintains self-renewal of BM-MSCs by inhibiting the expression of p27, p16, and p19 and alters the cell fate of BM-MSCs by enhancing osteoblast differentiation and inhibiting adipocyte differentiation at least in part by stimulating Sirt1 expression. Bmi-1 therefore plays a critical role in promoting osteogenesis. ß

To investigate the role of astroglial water channel aquaporin-4 (AQP4) in maintaining blood-brain barrier integrity, structure and permeability of the brain microvessels were investigated in adult AQP4 knockout mice. Altered ultrastructure of brain microvessels, including open tight junctions and swollen perivascular astrocytic endfeet, were frequently observed in the AQP4 null mice. Likewise, AQP4 deficiency significantly downregulated expression of glial fibrillary acidic protein in perivascular processes of astrocytes. Furthermore, the horseradish peroxidase analysis demonstrated hyperpermeability of the blood-brain barrier in AQP4 knockout mice. These findings provide direct evidence that AQP4 is essential for the maintenance of blood-brain barrier integrity.