A large number of macrophages and haematopoietic progenitor cells accumulate in pre-metastatic lungs in which chemoattractants, such as S100A8 and S100A9, are produced by distant primary tumours serving as metastatic soil. The exact mechanism by which these chemoattractants elicit cell accumulation is not known. Here, we show that serum amyloid A (SAA) 3, which is induced in pre-metastatic lungs by S100A8 and S100A9, has a role in the accumulation of myeloid cells and acts as a positive-feedback regulator for chemoattractant secretion. We also show that in lung endothelial cells and macrophages, Toll-like receptor (TLR) 4 acts as a functional receptor for SAA3 in the pre-metastatic phase. In our study, SAA3 stimulated NF-kappaB signalling in a TLR4-dependent manner and facilitated metastasis. This inflammation-like state accelerated the migration of primary tumour cells to lung tissues, but this was suppressed by the inhibition of either TLR4 or SAA3. Thus, blocking SAA3-TLR4 function in the pre-metastatic phase could prove to be an effective strategy for the prevention of pulmonary metastasis.

Flt-1, also known as vascular endothelial growth factor receptor 1 (VEGFR-1), is a high-affinity tyrosine kinase receptor for VEGF and is expressed almost exclusively on vascular endothelial cells. As an exception, Flt-1 transcript was recently found to be expressed in human peripheral blood monocytes. However, the protein of the Flt-1 receptor on the cell surface of monocytes is yet to be identified, and whether the Flt-1 protein is expressed during the differentiation of monocyte-macrophage lineage cells has not been examined. Using monoclonal antibodies against 2 different antigenic epitopes on the Flt-1 extracellular domain, this study found that the major population of the monocyte-marker CD97 ؉ cells in human peripheral blood express Flt-1 as a cell surface molecule. VEGFR-2 (KDR/Flk-1) was not expressed at detectable levels in these cells. An Flt-1 neutralizing monoclonal antibody significantly suppressed VEGF-induced migration of the monocytes, suggesting an important role for Flt-1 in the biologic function of monocytes. Furthermore, CD34 ؉ cells in human cord blood, originally negative for the Flt-1 expression, differentiated into Flt-1 ؉ cells in association with the appearance of monocyte-macrophage markers after a 2-week culture in the presence of hematopoietic cytokines. In addition, the Flt-1 ؉ CD11b ؉ cell fraction from CD34 ؉ cells was found to efficiently differentiate into multinuclear osteoclasts in the presence of macrophage colony-stimulating factor and osteoclast differentiation factor. These results strongly suggest that Flt-1 is a novel cell surface marker as well as a biologically functional molecule for monocyte-macrophage lineages in humans. IntroductionVascular endothelial growth factor (VEGF) and its receptors Flt-1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2) are well established as the essential regulatory system for blood vessel formation in embryogenesis. [1][2][3][4] Other VEGF family members such as VEGF-C and VEGF-D and their specific receptor Flt-4 (VEGFR-3) are also important for angiogenesis as well as lymphoangiogenesis in the embryo. 5 The specificity of VEGF and its receptor system for vascular endothelial cells appears to be based on at least 2 factors, the endothelial cell-specific expression of Flt-1 and KDR/Flk-1 6,7 and a unique signaling pathway from the receptors. We have recently shown that VEGF-dependent DNA synthesis in primary endothelial cells is mainly mediated via the phospholipase C␥-protein kinase C-mitogen-activated protein (MAP) kinase pathway, different from the signal transduction of the representative receptor tyrosine kinases such as EGF receptor. 8,9 Most of the signals for VEGF-induced cell proliferation and vascular permeability are considered to be mediated by KDR/ Flk-1, which has strong tyrosine kinase activity. [10][11][12] On the other hand, the Flt-1 bears an affinity for VEGF that is about 10-fold higher than KDR, but contains much weaker tyrosine kinase activity, suggesting a regulatory function in the VEGF signaling in endothelial cells. [13][14][...

Flk-1 (human counterpart, KDR) tyrosine kinase, which is one of the two VEGF receptors, is crucial for vascular development. Recently, we showed that, among tyrosine residues of KDR, tyrosine residues 1175 (Y1175, corresponding to Y1173 in murine Flk-1) and Y1214 (Y1212 in Flk-1) are autophosphorylated in response to VEGF, and that Y1175 is important for VEGF-dependent phospholipase C␥͞PKC͞mitogen-activated protein kinase activation leading to DNA synthesis in cultured endothelial cells. However, the importance of these tyrosine residues in Flk-1͞KDR in vivo is not yet known. To examine the role of these Flk-1 tyrosine residues in vivo, we generated knock-in mice substituting Y1173 and Y1212 of the Flk-1 gene with phenylalanine, respectively. As a result, Flk-1 1173F homozygous mice died between embryonic days 8.5 and 9.5 without any organized blood vessels or yolk sac blood islands, and hematopoietic progenitors were severely reduced, similar to the case of Flk-1 null mice. In contrast, Flk-1 1212F homozygous mice were viable and fertile. These results suggest that the signaling via Y1173 of Flk-1 is essential for endothelial and hematopoietic development during embryogenesis.Flk-1͞KDR ͉ single tyrosine-phenylalanine mutation ͉ tyrosine kinase receptor V EGF is essential for many angiogenic processes both in normal and pathological conditions (1, 2). VEGF binds two tyrosine kinase receptors, Flt-1 (also known as VEGF receptor 1) (3, 4) and Flk-1 (also known as VEGF receptor 2, KDR as human counterpart) (5-7) with high affinity. Flt-1-and Flk-1-deficient mice both die in utero between embryonic days (E) 8.5 and E9.5 but have different phenotypes. Flt-1-deficient embryos showed an overgrowth of endothelial cells and disorganization of blood vessels (8). Moreover, Flt-1 tyrosine kinase-deficient mice showed normal vascular development (9), suggesting that the Flt-1 extracellular domain acts as a negative regulator of VEGF, and that Flt-1 tyrosine kinase is not necessary for vasculogenesis during development. On the other hand, Flk-1-deficient mice lack both mature endothelial and hematopoietic cells, indicating that Flk-1 is crucial for vascular development (10).Ligand binding to Flk-1͞KDR induces autophosphorylation of Flk-1͞KDR intracellular tyrosine residues and activates several signaling pathways, leading to cell proliferation, survival, migration, and permeability (11). Recently, we have shown that tyrosine residues 1175 (Y1175) and Y1214, but not Y801, on KDR are highly autophosphorylated in response to VEGF, and that Y1175 is crucial for VEGF-dependent cell proliferation via the phospholipase C␥ (PLC␥)͞PKC͞mitogen-activated protein kinase (MAPK) pathway in cultured endothelial cells (12). However, the importance of these tyrosine residues in Flk-1͞KDR in vivo remains to be elucidated.To develop a pharmaceutical drug(s) that efficiently suppresses angiogenesis in diseases such as cancer, it is important to identify the tyrosine residue(s) that is involved in the critical signaling pathway via Flk-1͞KDR for ...