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.
Highlights
Macrophage-stimulating protein (MSP, known as hepatocyte growth factor–like protein, HGFL) is a plasminogen-like growth factor that mediates its biological activities by activating the receptor tyrosine kinase RON [recepteur d'origine nantais, known as macrophage stimulating receptor-1, (MSTR1)], a member of the MET proto-oncogene family [1]
In vitro activation of pro-MSP by recombinant hepsin Substrate profiling of hepsin by the use of a synthetic combinatorial library determined (P/K)-(K/Q)-(T/L/N)-R as the P4-P1 consensus sequence [19], which is in good agreement with the identified cleavage site sequences from macromolecular substrates of hepsin
The results showed that soluble form of hepsin (sHepsin) (EC50 1⁄4 2.4 Æ 0.3 nmol/L) was 5-fold and 7-fold more efficient than sMT-SP1 (EC50 1⁄4 11.7 Æ 1.3 nmol/L) and HGF activator (HGFA) (EC50 1⁄4 17.7 Æ 1.9 nmol/L), respectively
Summary
Macrophage-stimulating protein (MSP, known as hepatocyte growth factor–like protein, HGFL) is a plasminogen-like growth factor that mediates its biological activities by activating the receptor tyrosine kinase RON [recepteur d'origine nantais, known as macrophage stimulating receptor-1, (MSTR1)], a member of the MET proto-oncogene family [1]. Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). MSP is constitutively expressed by hepatic parenchymal cells, as well as in lungs, adrenal glands, placenta, kidney, and pancreas [4, 5]. It is secreted as an inactive single-chain precursor pro-MSP, which requires proteolytic cleavage at the Ser-Lys-Leu-Arg483#Val484 bond to attain functional activity [5, 6]. Unlike HGF, where the high-affinity MET-binding site resides in the a-chain, the high-affinity RON-binding site for MSP is located on the mature b-chain that forms following pro-MSP cleavage [10]. Several trypsin-like serine proteases, including MT-SP1 ( known as matriptase) and HGF activator (HGFA; refs. 11–15), are known to activate proMSP, leading to typical cellular responses mediated by the RON signaling pathway [11, 12]
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