Conserved expression of hepatocyte growth factor activator inhibitor type-2/placental bikunin in human colorectal carcinomas
Introduction
Hepatocyte growth factor (HGF) is a pleiotropic factor initially identified as a heparin-binding growth factor for hepatocytes [1], [2] indistinguishable from scatter factor (SF), a motility factor [3], [4], [5]. HGF/SF is a mesenchymally-derived glycoprotein secreted as an inactive precursor. Normally, it remains in this precursor form, probably associated with the extracellular matrix in the producing tissues [6]. To generate biologically active HGF/SF, the proteolytic conversion of the single-chain precursor form to the two-chain heterodimeric active form is essential [6], and this activation process is thus a critical event to regulate the HGF/SF activity in the extracellular microenvironment. HGF activator (HGFA) was identified as a serine proteinase having an efficient pro-HGF/SF activating activity [7], [8]. This enzyme is secreted mainly by the liver as an inactive zymogen (pro-HGFA), circulating in the blood in this form [8], [9], [10]. This zymogen can be activated by limited proteolysis brought about by thrombin in an injured tissue [10], [11], [12]. In fact, the pro-HGF/SF activation in the injured rat liver was abrogated by treatment with anti-HGFA antibody indicating that HGFA is crucially involved in the activation of pro-HGF/SF in vivo [10]. The activity of HGFA is not inhibited by major plasma proteinase inhibitors, and in fact, HGFA is active in serum [9].
Recently, two novel Kunitz-type serine proteinase inhibitors with potent inhibitory activity against HGFA were discovered. These inhibitors were designated as HGFA inhibitor type 1 and type 2 (HAI-1 and HAI-2) [13], [14]. cDNA cloning of HAI-2 revealed that this protein was identical to placental bikunin (PB) [15]. Subsequently it was also reported as a protein over-expressed in pancreatic cancer designated as kop [16]. HAI-2/PB has two Kunitz domains and a presumed transmembrane domain [14], [15], [16]. It inhibits not only HGFA [14], but also human plasmin, trypsin, tissue and plasma kallikreins, and factor XIa [17]. To a lesser degree it also inhibits factor IXa, factor Xa and factor XIIa [17]. Thus, although the exact pathophysiological role of HAI-2/PB in human tissues is undefined, it is reasonable to postulate that HAI-2/PB may have an important role in the regulation of HGF-induced tissue responses and of proteinase activities in various pathophysiological conditions including inflammation, coagulation, fibrinolysis and tumorigenesis.
A number of recent studies have indicated that HGF/SF and its specific receptor c-Met tyrosine kinase could have important roles in tumor progression [18], [19], [20], [21]. And the c-met gene is over-expressed in colorectal carcinomas [22]. Furthermore, roles of serine proteinases such as plasmin and tumor-associated trypsin in the tumor progression, particularly in invasion/metastasis events, have been well documented. These lines of evidence suggested that HAI-2/PB may have a role in the progression of tumors including colorectal carcinomas. We have previously reported that another HGFA inhibitor, HAI-1, was expressed in the colorectal mucosa and that its expression levels were decreased in colon carcinomas [23]. However, the expression of HAI-2/PB in normal and neoplastic colorectal mucosal tissues remains to be clarified. In this report, we examined the expression of the HAI-2/PB mRNA and protein in the normal and neoplastic colorectal mucosal tissues. We found that HAI-2/PB is consistently expressed in the normal colorectal mucosa and the expression is conserved in neoplastic differentiation.
Section snippets
Tissue collection and histological assessment
All tissue was received freshly from the operating room. The cases analyzed were 24 primary colorectal carcinomas. In each case, tissue samples were taken from the primary tumor and corresponding normal mucosal tissue, and were used for RNA extraction as described below. All colon pathology was assessed independently by two pathologists. Diagnoses and terminology were those in Histological Typing of Intestinal Tumors, World Health Organization. Sections taken adjacent to the tissue used for RNA
Results
RNA blot analysis revealed that HAI-2/PB mRNA was consistently expressed in the normal human colorectal mucosa. Fig. 1 shows representative RNA blot results for 12 samples of human colorectum. A single major HAI-2 transcript of 1.6 kb was identified in all cases. The expression was also consistently observed in the corresponding neoplastic tissues (Fig. 1). The level of each signal, normalized by division through the corresponding G3PDH signal, was quantified, and a ratio of the signal in the
Discussion
In the present study, we found that HAI-2/PB was consistently expressed in the human normal colonic mucosa, this expression being significantly conserved in the neoplastic differentiation. An immunohistochemical study also supported the results obtained by RNA blot analyses. In contrast to HAI-1, another HGFA inhibitor which is expressed on the basolateral surface of the epithelial cells [13], [23], [24], the immunoreactivity of HAI-2/PB was predominantly intracytoplasmic and occasionally on
Acknowledgements
We are grateful to Miss. N. Iwakiri for preparation of histological sections. This work was partly supported by Osaka Cancer Research Foundation (H.I.).
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2000, Journal of Biological ChemistryCitation Excerpt :Another possibility is that a Kunitz-containing part of HAI-2/PB is not exposed to the extracellular milieu but is exposed to a lumen of a vesicular compartment depending on its post-Golgi trafficking pathway. Although convincing evidence supporting this possibility is lacking at present, our previous immunohistochemical study showed that HAI-2/PB was preferentially stained intracellularly in the epithelial cells, whereas HAI-1 was stained on the basolateral cellular surface (14, 15, 25, 26). Alternatively, HAI-2/PB may be very quickly released from the cell surface.