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Hepatocyte growth factor activator inhibitor type 2 (HAI-2) is a serine proteinase inhibitor containing two Kunitz-type inhibitor domains, initially identified as a potent inhibitor of hepatocyte growth factor activator (HGFA). In a previous study (Biochem. Biophys. Res. Commun. 255, 740-748, 1999), we reported that a predominant transcript of mouse HAI-2 is a splicing variant lacking the first Kunitz domain (KD-1). Since KD-1 was reported to be responsible for the inhibition of HGFA in human HAI-2 and the second Kunitz domain (KD-2) of human HAI-2 was much less inhibitory against HGFA, it has been suggested that most of mouse HAI-2 may be ineffective in inhibiting HGFA. In this study, we have performed functional characterization of Kunitz domains in mouse HAI-2 by using recombinant proteins synthesized by Chinese hamster ovary cells without or with point mutation in the putative reactive site of each Kunitz domain. The results revealed that, unlike human HAI-2, KD-2 of mouse HAI-2 efficiently inhibits HGFA. Therefore, the major mouse HAI-2 protein that consists only of KD-2 can be a potent inhibitor of HGF activation in vivo.

Hepatocyte growth factor (HGF) activator inhibitor type 1 (HAI-1) and type 2 (HAI-2) were recently discovered as specific inhibitors of HGF activator. Each of them contains two Kunitz-type serine protease inhibitor domains and a transmembrane domain, so that their overall structures are similar to each other. In this study, mouse genes encoding HAI-1 and HAI-2 were cloned by screening of a mouse genomic bacterial artificial chromosome library and by polymerase chain reaction of mouse genomic DNA, respectively. The genes (mHAI-1 and mHAI-2) were defined to consist of 11 and eight exons spanning 11 kbp and 9.5 kbp, respectively. Neither a TATA nor CAAT box was found in 5′-flanking regions of both genes and no apparent homologous portion was observed between mHAI-1 and mHAI-2 promoter regions. Promoter assay of mHAI-1 and human HAI-1 revealed that the potential binding sites of a complex of Egr-1–3 and Sp1, which was well-conserved between human (−42 to −58) and mouse (−44 to −57), might be a key portion of its transcriptional regulation to function as not only house-keeping but also early responsive genes.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) and type 2 (HAI-2) are recently discovered Kunitz-type serine protease inhibitors which can be purified and cloned from human stomach cancer cell line MKN45 as specific inhibitors against hepatocyte growth factor activator (HGFA). HAI-2 was identical with the protein originally reported as placental bikunin. Both proteins contain two Kunitz inhibitor domains (KDs), of which the first domain (KD1) is mainly responsible for the inhibitory activity against HGFA, and are expressed ubiquitously in various tissues. In this study, we cloned the genes coding for these two structurally similar proteins by screening of human genomic bacterial artificial chromosome (BAC) library and their genomic structures were compared. HAI-1 and -2 genes consist of 11 and 8 exons spanning 12 kbp and 12.5 kbp, respectively. Three exons were inserted between KD1 and KD2 of each gene, of which the middle one was the low-density lipoprotein (LDL) receptor-like domain (HAI-1) and the testis specific exon (HAI-2). Apparently homologous regions between HAI-1 and -2 were not found in 5'-flanking region and neither TATA nor CAAT box was present. The genes were mapped to chromosome 15q15 (HAI-1) and 19q13.11 (HAI-2). These results suggested that although HAI-1 and -2 genes might be derived from same ancestor gene, they acquired distinctive in vivo roles during their evolution.

A cDNA encoding mouse hepatocyte growth factor activator (HGFA) has been cloned by RT-PCR, based on the screening result from the database of expressed sequence tags. Subsequently, its gene was cloned from a mouse genomic bacterial artificial chromosome library using the cDNA as a probe. Sequencing analysis revealed that mouse HGFA protein deduced from the cDNA, similar to its human and rat counterparts, has two epidermal growth factor-like domains, type 1 and 2 fibronectin homology domains, a single kringle domain and a catalytic domain of serine proteinase, and the gene consists of 14 exon spanning approximately 7.5 kb. Interestingly, mouse HGFA mRNA was detected not only in the liver but also in the gastrointestinal tract by RNA blot analysis. Since hepatocyte growth factor (HGF) is up-regulated in the damaged gastrointestinal mucosa, our present data suggest that HGFA might activate proHGF directly in the gastrointestinal mucosa and play an important role in wound repair throughout the gastrointestinal tract.

Hepatocyte growth factor (HGF) activator inhibitor type 1 (HAI-1) and type 2 (HAI-2) are new Kunitz-type serine protease inhibitors that were recently purified and cloned from the human stomach cancer cell line MKN45 as specific inhibitors against HGF activator. Both proteins contain two Kunitz inhibitor domains and are expressed abundantly throughout the gastrointestinal tract, in addition to the placenta, pancreas, and kidney. In this study, to assess the possible roles of HAI-1 and HAI-2 in the intestinal mucosa, we examined the expression of HAI-1 and HAI-2 during regeneration of the intestinal mucosa. Immunohistochemical studies revealed that HAI-1 but not HAI-2 was detected more strongly in regenerative epithelium than in normal epithelium, although both proteins were detected throughout the human gastrointestinal tract. During the course of acetic acid-induced experimental colitis in an in vivo mouse model, HAI-1 but not HAI-2 was upregulated in the recovery phase, suggesting that HAI-1 but not HAI-2 is associated with the regeneration of damaged colonic mucosa. Upregulation of HAI-1 may serve to downregulate the proliferative response after initial activation of MET receptor by HGF/scatter factor after an injury.

A cell line designated NYGM was established from a human cerebral glioblastoma multiforme (GBM) obtained from a 75-year-old Japanese woman. The cell line has grown slowly without interruption and has been propagated continuously by serial passages (more than 80 passage) during the past 3 years. The cultured cells were fusiform or polyhedral in shape. The population doubling time was 24 hours. The chromosomal number varied between 77 and 88, with modal chromosomal number of 84. NYGM cells concomitantly expressed MET receptor tyrosine kinase (a product of c-met protooncogene) and its ligand HGF/SF (hepatocyte growth factor/scatter factor), as well as HGF activator and HGF activator inhibitors. The cells might be useful for the study of pericellular regulation of HGF/SF-MET signaling and HGF activation of GBM cells.

Activation of hepatocyte growth factor (HGF) is a crucial limiting step in HGF-induced signaling pathway. The HGF activator inhibitor type 1 (HAI-1) was identified as a potent inhibitor of HGF activator (HGFA), a serine proteinase that is responsible for the activation of HGF in vivo. HAI-1 is an integral membrane Kunitz-type serine proteinase inhibitor, and its mRNA has been reported to be most abundant in the placenta. In this report, specific antibody to HAI-1 was used in an immunohistochemical procedure to determine the localization of HAI-1 in human placenta. HAI-1 was expressed in cytotrophoblasts (Langhans' cells) of the double-layered trophoblastic epithelium of chorionic villi tissue, and syncytiotrophoblasts were almost negative. On the other hand, extravillous trophoblasts of cytotrophoblastic columns showed markedly decreased immunoreactivity, and those infiltrating into the superficial decidua membrane of early placenta were hardly stainable. The amnionic epithelial cells were also immunostained intensely. The presence of HAI-1 mRNA was also confirmed in a cultured human cytotrophoblastic cell line. In addition to HAI-1, low but distinct expression of HGFA mRNA was observed in the placenta tissue and cultured cytotrophoblasts by using a sensitive RT-PCR method. Since HGF plays an essential role in the placenta development, expression of HAI-1 and HGFA may have an important regulatory role in the placenta. The localization of HAI-1 in the proliferating trophoblastic stem cells (Langhans' cells), but not in syncytiotrophoblasts and extravillous trophoblasts, suggest a possible role of HAI-1 in the proliferation of trophoblasts.

Hepatocyte growth factor activator (HGFA) is responsible for proteolytic activation of the precursor form of hepatocyte growth factor in injured tissues. To date, two specific inhibitors of HGFA have been identified, namely HGFA inhibitor type 1 (HAI-1) and type 2 (HAI-2)/placental bikunin (PB). Both inhibitors are first synthesized as integral membrane proteins having two Kunitz domains and a transmembrane domain, and are subsequently released from cell surface by shedding. Here we show that an active form of HGFA is specifically complexed with membrane-form HAI-1, but not with HAI-2/PB, on the surface of epithelial cells expressing both inhibitors. This binding required the enzyme activity of HGFA. The selective binding of HGFA to the cell surface HAI-1 was further confirmed in an engineered system using Chinese hamster ovary cells, in which only the cells expressing HAI-1 retained exogenous HGFA. The binding of HGFA to HAI-1 was reversible, and no irreversible modifications affecting the enzyme activity occurred during the binding. Importantly, HAI-1 and the HGFA.HAI-1 complex were quickly released from the cell surface by treatment with phorbol 12-myristate 13-acetate or interleukin 1beta accompanying the generation of 58-kDa fragments of HAI-1, which are less potent against HGFA, as well as significant recovery of HGFA activity in the culture supernatant. This regulated shedding was completely inhibited by BB3103, a synthetic zinc-metalloproteinase inhibitor. We conclude that HAI-1 is not only an inhibitor but also a specific acceptor of active HGFA, acting as a reservoir of this enzyme on the cell surface. The latter property appears to ensure the concentrated pericellular HGFA activity in certain cellular conditions, such as tissue injury and inflammation, via the up-regulated shedding of HGFA.HAI-1 complex. These findings shed light on a novel function of the integral membrane Kunitz-type inhibitor in the regulation of pericellular proteinase activity.