In Vitro Evidence That KLK14 Regulates the Components of the HGF/Met Axis, Pro-HGF and HGF-Activator Inhibitor 1A and 1B
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Biol. Chem. 2016; 397(12): 1299–1305 Short Communication Janet C. Reid, Nigel C. Bennett, Carson R. Stephens, Melanie L. Carroll, Viktor Magdolen, Judith A. Clements and John D. Hooper* In vitro evidence that KLK14 regulates the components of the HGF/Met axis, pro-HGF and HGF-activator inhibitor 1A and 1B DOI 10.1515/hsz-2016-0163 Received April 1, 2016; accepted August 10, 2016; previously Kallikrein-related peptidase (KLK) 14 (Hooper et al., published online August 17, 2016 2001; Yousef et al., 2001) is one of the 15 members of the human KLK family of trypsin-fold serine proteases Abstract: Kallikrein-related peptidase (KLK) 14 is a serine which also includes the prostate cancer biomarker KLK3 protease linked to several pathologies including prostate (or prostate-specific antigen; PSA) (Dong et al., 2014). cancer. We show that KLK14 has biphasic effects in vitro KLK14 spans 267 residues incorporating a signal peptide, on activating and inhibiting components of the pros- that instructs for secretion, and a short pro-sequence tate cancer associated hepatocyte growth factor (HGF)/ that is cleaved at Lys40-Ile41 to generate the catalyti- Met system. At 5–10 nm, KLK14 converts pro-HGF to cally active protease. Mature KLK14 has trypsin-like the two-chain heterodimer required for Met activation, activity with preference for cleavage after Arg (Brattsand while higher concentrations degrade the HGF α-chain. et al., 2005; Felber et al., 2005; Oikonomopoulou et al., HGF activator-inhibitor (HAI)-1A and HAI-1B, which 2006; Stefansson et al., 2006; Borgono et al., 2007b; inhibit pro-HGF activators, are degraded by KLK14 when Rajapakse and Takahashi, 2007; de Veer et al., 2012) protease:inhibitor stoichiometry is 1:1 or the protease is and there is also evidence for low-level chymotrypsin- in excess. When inhibitors are in excess, KLK14 generates like activity (Brattsand et al., 2005; Felber et al., 2005). HAI-1A and HAI-1B fragments known to inhibit pro-HGF KLK14 is a positive regulator of the proteolytic cascade activating serine proteases. These in vitro data suggest that mediates skin desquamation, and the absence of that increased KLK14 activity could contribute at multi- its inhibition is implicated in the skin barrier disorder ple levels to HGF/Met-mediated processes in prostate and Netherton syndrome (Prassas et al., 2015). In addition, other cancers. serum KLK14 levels are significantly elevated in pros- tate cancer patients compared with healthy men, and Keywords: hepatocyte growth factor; HGF inhibitor; tissue levels correlate with prostate tumor status and kallikrein-related peptidase 14; Met; prostate cancer. are associated with disease progression defined by PSA relapse (Borgono et al., 2007b; Rabien et al., 2008). A functional link between increased KLK14 expression *Corresponding author: John D. Hooper, Mater Research Institute – and prostate cancer is suggested by its regulation of University of Queensland, Woolloongabba, Queensland 4102, proteins that modulate progression of this malignancy. Australia, e-mail: [email protected] For example, KLK14 cleaves insulin-like growth factor Janet C. Reid: Mater Research Institute – University of Queensland, binding proteins, thereby increasing the availability of Woolloongabba, Queensland 4102, Australia; and Institute of insulin-like growth factor (IGF) to promote cell prolifera- Health and Biomedical Innovation, Translational Research Institute, Queensland University of Technology, Woolloongabba, Queensland tion (Borgono et al., 2007b). In addition, KLK14 modifies 4102, Australia the prostate cancer microenvironment via degradation Nigel C. Bennett, Carson R. Stephens , Melanie L. Carroll of the androgen transporter sex hormone-binding globu- and Judith A. Clements: Institute of Health and Biomedical lin (Sanchez et al., 2012), disruption of cell-cell contacts Innovation, Translational Research Institute, Queensland University via cleavage of desmoglein (Borgono et al., 2007a), and of Technology, Woolloongabba, Queensland 4102, Australia Viktor Magdolen: Klinische Forschergruppe der Frauenklinik der degradation of extracellular matrix proteins including Technischen Universität München, Klinikum rechts der Isar, Munich, collagens, laminin, fibronectin and vitronectin (Borgono Germany et al., 2007b; Rajapakse and Takahashi, 2007). Brought to you by | EP Ipswich Authenticated Download Date | 11/9/16 5:27 AM 1300 J.C. Reid et al.: KLK14 regulates pro-HGF, HAI-1A and HAI-1B Hepatocyte growth factor (HGF) mediates cell the most poorly differentiated tumors exhibiting the growth, cell motility and morphogenesis in numerous lowest levels of expression (Bergum and List, 2010; Tsai cell and tissue types via binding to the Met tyrosine et al., 2014). kinase receptor (Cecchi et al., 2012). In prostate cancer The overlapping elevated expression of KLK14 and HGF is abundantly expressed by stromal cells present HGF in prostate cancer, and the specificity that KLK14 has within the tumor microenvironment and serum levels for cleavage after Arg residues, which potentially includes are a strong predictor of metastasis to lymph nodes and the canonical activation site of pro-HGF (Arg494-Val495), disease recurrence (Gupta et al., 2008; Yasuda et al., prompted us to explore the role of KLK14 in the regula- 2009; Varkaris et al., 2011). Met is overexpressed by tion of the HGF/Met system. For this purpose we gener- malignant cells in primary tumors and is associated ated and purified recombinant KLK14 from insect cells, with high Gleason grade and tumor vascularization, first characterizing its enzymatic properties, then evalu- and potentially tumor stage (Nakashiro et al., 2003; ating its impact in vitro on pro-HGF and the inhibitors Strohmeyer et al., 2004; Jacobsen et al., 2013). Thus, par- HAI-1A and HAI-1B. To generate a suitable KLK14 expres- acrine signaling via the actions of stroma-produced HGF sion construct, the previously cloned coding sequence on the Met receptor, which is elevated on the surface of (Hooper et al., 2001), including the signal and pro-region malignant cells, is thought to be important in prostate sequence, was cloned in-frame in a pIB/V5-His expres- cancer progression and metastasis (Trusolino et al., sion vector (Invitrogen, Life Technologies Australia Pty 2010). HGF is produced as a pro-factor requiring activa- Ltd, Mulgrave, Victoria, Australia) generating KLK14 with tion to generate a two-chain heterodimer that includes a carboxyl-terminal V5 tag (GKPIPNPLLGLDST) and six an α-chain, containing four kringle domains, and a β- histidine residues. Insect Spodoptera frugiperda Sf9 cells chain that has homology to trypsin-fold serine proteases. (Gibco, Life Technologies, Mulgrave, Victoria, Australia) Activation occurs at Arg494-Val495 and is mediated by were stably transfected with the KLK14-V5-His construct the circulating Arg/Lys-specific trypsin-fold serine pro- and KLK14 was purified from the conditioned media using tease HGF-activator (HGFA) (Miyazawa, 2010) and the Ni-NTA Superflow resin (Qiagen, Doncaster, Victoria, plasma membrane serine proteases matriptase (Lee Australia) eluting in the manufacturer’s supplied elution et al., 2000; Kirchhofer et al., 2003) and hepsin (Herter buffer (Figure 1A). Pooled fractions of purified recombi- et al., 2005; Kirchhofer et al., 2005). The actions of these nant KLK14 were concentrated and dialyzed at 4°C against serine proteases in initiation of the HGF/Met signaling 5 mm NaH2PO4, 95 mm Na2HPO4, 0.01% (v/v) Tween20, axis are commonly negatively regulated by the Kunitz- pH 8.0 (Borgono et al., 2007b). Activation was then per- type inhibitors HGFA-inhibitor (HAI)-1, which has two formed by incubating KLK14 with the metalloprotease variants HAI-1A and HAI-1B, and HAI-2 (Kawaguchi and thermolysin (Calbiochem, San Diego, CA, USA) in the Kataoka, 2014). These inhibitors block proteolysis via ratio 1:12.5 (thermolysin:KLK4) for 90 min at 37°C. Activa- tight association, as a pseudo-substrate, with the active tion was apparent from the reduction in molecular weight site of the serine protease (Shia et al., 2005). The roles of KLK14 of ~4 kDa as a result of loss of the pro-domain of HAI-1 and HAI-2 in prostate cancer are not yet well (Figure 1B). For comparison we employed thermolysin- defined and recent expression studies suggest poten- activated recombinant KLK4, generated and purified as tially disparate roles for these inhibitors. For example, previously described (Ramsay et al., 2008), and bovine in contrast with the usual paradigm that reduced pro- trypsin ( Worthington Biochemical, Lakewood, NJ, USA), tease inhibitor levels contribute to protease-mediated or the soluble auto-activating form of hepsin lacking processes in cancer progression, HAI-1 protein levels are its intracellular and transmembrane domains (Kirch- higher in serum of patients with prostate cancer com- hofer et al., 2005) (generously provided by Dr. Daniel pared with men who have benign prostatic hyperplasia, Kirchhofer, Genentech Inc., San Francisco, CA, USA). and are also elevated in men who have metastasis and The proportion of active KLK4, KLK14, trypsin and hepsin castrate resistant prostate cancer compared to patients present in relevant assays was determined by incubat- with organ-confined disease (Nagakawa et al., 2006). ing protease (300 nm) with the suicide pseudo-substrate Similar changes were reported in prostate tumors with 4-methylumbelliferyl 4-guanidinobenzoate (MUGB; 1 μm) HAI-1 protein levels higher in patients with prostate (Jameson