Endogenous Ligand-Induced Activation of TLR4 in Pre-metastatic Phase Is Both Downstream and Upstream of TNF Signaling

Abstract

Recently accumulating information suggests the existence of endogenous TLR4 candidate including hyaluronan fragments, HMGB1, Tenascin, saturated fatty acids, S100A8, etc. (reviewed in [1]). We have added one more candidate SAA3 (serum amyloid A3) [2]. In our experimental metastasis assay, we subcutaneously inject tumor cells that never reach the lungs, which we call pre-metastatic lungs (pre-metastatic phase) [3]. Then we purposely inject labeled tumor cells via the tail vein and count their numbers in the lungs (metastatic phase). A cDNA microarray screening for up-regulated genes in pre-metastatic lungs between tumor-bearing and non-bearing mice gave both S100A8 and SAA3 in the top 50 genes [2, 4]. Surface plasmon resonance analysis of TLR4/MD-2 complex purified from baculovirus and S100A8 or SAA3 purified from mammalian cells provided direct evidence for binding. TLR is a well-studied pattern recognition receptor working as a sensor for bacterial endotoxin or lipopolysaccharide (LPS). S100A8 was initially identified in the synovial fluid of rheumatoid arthritis patients and is one of the members of well-conserved EF-hand Ca2+-binding protein family [5, 6]. SAA3 belong to the SAA family of acute phase reactant [7]. SAA1 and SAA2 are generated in liver in response to inflammation and integrated into HDL (high density lipoprotein). SAA3 is produced extra-hepatically, such as in myeloid cells and endothelial cells. We have shown that S100A8 induces SAA3 expression with promoter activation by two- to fourfolds [2]