In addition, RIP2 2/2 mice have increased bacterial burden and decreased IL-17A production in the lung

f by which Prx1 regulates VEGF is undefined. VEGF expression is elevated in hypoxic conditions. Elevated VEGF expression is a result of increased transcriptional activity that is mediated by the transcription factor HIF-1 . HIF-1 is comprised of two subunits, a constitutively expressed b subunit and an inducible labile a subunit. In non-inflammatory, normoxic conditions HIF-1a is associated with prolyl hydroxylases and von Hippel-Lindau -E3 ligase complexes that cause rapid proteosome mediated degradation of HIF-1a. In settings of reduced oxygen PHD activity is reduced allowing for translocation of HIF-1a to the nucleus where it interacts with HIF-1b and binds to hypoxia-response elements to simulate target gene Prx1 Activation of HIF-1 transcription. Several studies have shown that pro-inflammatory cytokines and TLR signaling can induce HIF-1a gene expression and increase HIF-1a stability in normoxic conditions. Lipopolysaccharide, the prototypical exogenous TLR4 ligand, can induce VEGF and HIF-1a gene expression via activation of NF-kB in macrophages, and dendritic cells. Other studies have demonstrated that generation of reactive oxygen rather than NF-kB is responsible for TLR4 mediated increases in VEGF and HIF-1a mRNA and protein levels in macrophages. Finally some studies indicate that the LPS:TLR4 interaction alone is insufficient for induction of VEGF and HIF-1a and requires simultaneous activation of the adenosine A2A receptor. Thus the mechanism of induction of VEGF by TLR4 agonists is somewhat controversial and warrants additional study. Activation of VEGF expression by TLR4 may be cell type specific. For example, LPS:TLR4 interaction induces VEGF expression in fibroblasts without increasing expression of HIF-1a, while induction in macrophages is dependent upon HIF-1a. The tumor microenvironment consists of tumor cells, extracellular matrix and host cell populations, including TG100 115 endothelial cells and their precursors, pericytes, muscle cells, fibroblasts and inflammatory cells. Previous study suggested that Prx1 stimulation of VEGF expression from host cells was important for angiogenesis. In this study we examined the mechanism by which Prx1 stimulates VEGF expression and migration in endothelial cells. We demonstrate that Prx1 regulates endothelial cell VEGF protein expression, mRNA levels, and promoter activity in a TLR4 signaling dependent manner. Analysis of VEGF promoter activity revealed that Prx1 induction of VEGF was dependent upon HIF-1 interaction with the hypoxic response element within the VEGF promoter. Subsequent studies showed that Prx1 and TLR4 interaction increases HIF-1a mRNA expression, promoter activity and protein levels in a NF-kB dependent manner. Additionally, Prx1 and TLR4 interaction leads to a MEK dependent eIF-4E phosphorylation, which may mediate increases in transcription and/or translation of HIF-1a. Interestingly, tumors with reduced Prx1 levels express lower levels of HIF-1a. Prx1 stimulation of VEGF expression results in endothelial cell proliferation and motility that is dependent upon TLR4, NF-kB, and HIF-1. Results Prx1 Stimulates Endothelial Cell Expression of VEGF The effect of rPrx1 on endothelial cell expression of VEGF protein and mRNA expression was tested in vitro. rPrx1 stimulated maximal VEGF expression in 2H-11 murine endothelial cell lysates within an hour. VEGF expression declined to baseline 4 hours after stimulation. VEGF secretion increased within 2 hours of stimulation. Leve