Angiogenesis, Metastasis, and the Cellular Microenvironment Proteolytic Activation of Pro-Macrophage-Stimulating Protein by Hepsin

Macrophage-stimulating protein (MSP) is a plasminogen-related growth factor and ligand for the receptor tyrosine kinase RON. TheMSP/RON system promotes wound healing and invasive tumor growth and suppresses proinflammatory immune response. MSP binding to RON requires proteolytic conversion of the inactive singlechain form (pro-MSP) into the disulfide-linked a/b heterodimer. The pro-MSP cleavage sequence (Ser-Lys-LeuArg#Val) closely matches the substrate recognition sequences of hepsin, a type II transmembrane serine protease, that is overexpressed in several cancers. Here, we show that recombinant hepsin cleaves pro-MSP at the consensus site Arg-Val with superior efficiency compared with the known activators MT-SP1 and hepatocyte growth factor activator (HGFA). At least 50% of pro-MSP was processed within 1 hour at a hepsin concentration of 2.4 nmol/L and at a molar enzyme to substrate ratio of 1:500. An uncleavable single-chain variant of MSP weakly bound to a RON–Fc fusion protein, whereas hepsin-cleaved MSP bound with a KD of 10.3 nmol/ L, suggesting that the high-affinity binding site in MSP b-chain was properly formed. LNCaP prostate cancer cells overexpressing hepsin on the cell surface efficiently activated pro-MSP, which was blocked by a specific anti-hepsin antibody. Incubation of pro-MSP with hepsin led to robust RON-mediated phosphorylation of mitogen-activated protein kinase, ribosomal S6 protein, and Akt in human A2780 ovarian carcinoma cells stably expressing RON protein. In macrophages, pro-MSP with hepsin induced chemotaxis and attenuated lipopolysaccharide-dependent production of nitric oxide. These findings suggest that the MSP/RON signaling pathway may be regulated by hepsin in tissue homeostasis and in disease pathologies, such as in cancer and immune disorders.Mol Cancer Res; 9(9); 1175–86. 2011 AACR.