Loss of membrane-bound serine protease inhibitor HAI-1 induces oral squamous cell carcinoma cells' invasiveness.

A loss of balance between cell membrane-associated proteases and their inhibitors may underlie cancer invasion and metastasis. We analysed the roles of a membrane- associated serine protease inhibitor, HAI-1, in oral squamous cell carcinoma (OSCC). While membranous HAI-1 was widely observed in cancer cells of human OSCC tissues, this was significantly reduced at the infiltrative invasion front. In vitro, HAI-1 was detected in all eight OSCC cell lines examined, in which its cognate membrane protease, matriptase was also expressed. HAI-1 expression knock-down (KD) in OSCC lines, SAS and HSC-3, reduced the growth of both lines in vitro but significantly enhanced SAS tumourigenicity in vivo, which was accompanied by histological changes suggestive of the epithelial-mesenchymal transition. Both HAI-1-KD lines also exhibited significantly enhanced migratory capability, and membrane-associated but not truncated HAI-1 was required to rescue this phenotype. Other OSCC lines (HSC-2, Sa3, Ca9-22) also showed enhanced migration in response to HAI-1 KD. The enhanced migration is partly attributed to dysregulation of matriptase, as simultaneous matriptase KD alleviated the migration of HAI-1-KD cells. HAI-1 deficiency also altered the expression of CD24, S100A4, CCND2 and DUSP6, all of which are involved in tumour progression. While matriptase was involved in the increased CD24 expression associated with HAI-1 deficiency, the protease appeared to be not responsible for the altered expression of other genes. Therefore, a matriptase-independent mechanism for the invasiveness associated with HAI-1 KD is also present. Together, these observations suggest that HAI-1 has a crucial suppressive role in OSCC cell invasiveness.

Activation of macrophage-stimulating protein by human airway trypsin-like protease.

Macrophage-stimulating protein (MSP) circulates as a proform protein and requires proteolytic processing for activation. Respiratory ciliated cells express the MSP receptor, recepteur d'origine nantais (RON), at the apical surface, which reportedly has an important role in ciliary function. Like RON, human airway trypsin-like protease (HAT) is also expressed at the apical surface of ciliated cells. Here we show that HAT cleaves proMSP at the physiological activation site, Arg483-Val484. MSP processed by HAT could induce chemotactic responses and morphological changes of peritoneal macrophages. In human respiratory epithelial cells, knock down of HAT expression reduced proMSP processing and RON autophosphorylation. We suggest that HAT is important for MSP-RON signaling in the respiratory tract.

Hepatocyte growth factor activator is a serum activator of single-chain precursor macrophage-stimulating protein.

Macrophage-stimulating protein (MSP) is a plasma protein that circulates as a single-chain proform. It acquires biological activity after proteolytic cleavage at the Arg483-Val484 bond, a process in which serum and cell surface serine proteinases have been implicated. In this article, we report that hepatocyte growth factor activator (HGFA), a serum proteinase which activates hepatocyte growth factor in response to tissue injury, may have a critical role in the activation of pro-MSP. In vitro analysis has revealed that human HGFA efficiently cleaves human pro-MSP at the physiological activation site without further degradation, resulting in biologically active MSP, as measured by the chemotactic response and MSP-induced morphological change of peritoneal macrophages. The processing of pro-MSP by HGFA is 10-fold more efficient than processing by factor XIa. To search for a role of HGFA in pro-MSP activation, we analyzed the processing of mouse pro-MSP in sera from HGFA-knockout (HGFA(-/-)) mice. The proform of MSP was the predominant molecular form in the plasma of both wild-type and HGFA(-/-) mice. In wild-type sera, endogenous pro-MSP was progressively converted to the mature two-chain form during incubation at 37 degrees C. However, this conversion was significantly impaired in sera from HGFA(-/-) mice. The addition of recombinant HGFA to HGFA-deficient serum restored pro-MSP convertase activity in a dose-dependent manner, and a neutralizing antibody to HGFA significantly reduced the conversion of pro-MSP in wild-type serum. Moreover, initial infiltration of macrophages into the site of mechanical skin injury was delayed in HGFA(-/-) mice. We suggest that HGFA is a major serum activator of pro-MSP.