title: Proteolytic activation of pro-macrophage-stimulating protein by hepsin.
abstract: Macrophage-stimulating protein (MSP) is a plasminogen-related growth factor and ligand for the receptor tyrosine kinase RON. The MSP/RON system promotes wound healing and invasive tumor growth and suppresses proinflammatory immune response. MSP binding to RON requires proteolytic conversion of the inactive single-chain form (pro-MSP) into the disulfide-linked α/β heterodimer. The pro-MSP cleavage sequence (Ser-Lys-Leu-Arg(483)↓Val(484)) 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(483)-Val(484) 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 K(D) of 10.3 nmol/L, suggesting that the high-affinity binding site in MSP β-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.
authors.affiliation: Department of Early Discovery Biochemistry, Genentech, Inc., 1 DNA Way, MS #27, South San Francisco, CA 94080, USA.
authors.forename: Rajkumar
authors.initials: R
authors.lastname: Ganesan
authors.forename: Ganesh A
authors.initials: GA
authors.lastname: Kolumam
authors.forename: S Jack
authors.initials: SJ
authors.lastname: Lin
authors.forename: Ming-Hong
authors.initials: MH
authors.lastname: Xie
authors.forename: Lydia
authors.initials: L
authors.lastname: Santell
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Wu
authors.forename: Robert A
authors.initials: RA
authors.lastname: Lazarus
authors.forename: Amitabha
authors.initials: A
authors.lastname: Chaudhuri
authors.forename: Daniel
authors.initials: D
authors.lastname: Kirchhofer
mesh_terms: D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D008264:Macrophages / Q000378:metabolism; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010051:Ovarian Neoplasms / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000378:metabolism*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C430890:macrophage stimulating protein, precursor, human; C080835:RON protein; D020794:Receptor Protein-Tyrosine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases; C081092:HGF activator; D012697:Serine Endopeptidases; C055645:hepsin