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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.

Hepatocyte growth factor/scatter factor (HGF/SF) contributes to the malignant progression of human gliomas. We investigated the effect of HGF/SF on matrix metalloproteinase-2 (MMP-2), membrane type 1 matrix metalloproteinase (MT1-MMP) and tissue inhibitors of metalloproteinases (TIMPs), expressions of c-Met/HGF receptor-positive human glioblastoma cells. Treatment of U251 human glioblastoma cells with HGF/SF resulted in enhanced secretion of MMP-2 with an increased level of the active form. This was accompanied by enhanced expression (2.5-fold) of mRNA specific for MMP-2. The stimulatory effect of HGF/SF on MMP-2 expression did not occur in the presence of herbimycin A, a protein tyrosine kinase inhibitor. MT1-MMP, a cell-surface activator of proMMP-2, was also up-regulated by HGF/SF in a dose-dependent manner. By contrast, the level of TIMP-1 mRNAs was not altered significantly and that of TIMP-2 was reduced mildly by the HGF/SF treatment, suggesting that HGF/SF may eventually modulate a balance between MMP-2 and TIMPs in favor of the proteinase activity in the glioma cell microenvironment. HGF/SF also stimulated MMP-2 expression of other glioblastoma cell lines. Since glioblastomas frequently co-express HGF/SF and its receptor, our results suggest that HGF/SF might contribute to the invasiveness of glioblastoma cells through autocrine induction of MMP-2 expression and activation. Int. J. Cancer 82:274–281, 1999. © 1999 Wiley-Liss, Inc.