Figure 1.

Role of HAI-2 in repressing matriptase activation and prostate cancer cell motility. (A) Examination of CWR22Rv1, 103E and N2 cell migration and invasion using transwell assays. Cells were seeded at a density of 2 × 10⁵ cells per well into a Boyden chamber coated with or without Matrigel. Regular culture medium containing 10% FBS was used as a chemoattractant in lower wells. The periods of incubation time were 24 hours for migration assays and 48 hours for invasion assays. The migrating and invasive cells on the bottoms of Boyden chamber were stained with 1% crystal violet and imaged by a microscopy (magnification, ×100). Three independent experiments were performed and a set of representative images were shown. (B) Quantification of the migrating and invasive cells in Fig. 1A by ImageJ software. The data were statistically calculated from of three independent experiments and presented as mean ± SD. (**p < 0.01; ***p < 0.001, one-way ANOVA). (C) The activation process of matriptase and the schematic structure of matriptase and HAI-1 on plasma membrane. Upon activation, latent matriptase (zymogen, 70 kDa) can undergo a cleavage process at the residue of R614 by autoactivation, androgen-induced TMPRSS2⁴⁷, COX-2⁹⁹ or ErbB-2 signaling⁴⁵, to become active and immediately form a complex with HAI-1 (120 kDa). The activated matriptase-HAI-1 complexes were used to generate monoclonal antibodies and two monoclonal antibodies (M32 and M69) were used in this study. M32 can recognize the third LDLR domain of matriptase, and M69 can specifically interact with the activated protease domain of matriptase¹⁰⁰, as shown in the figure. Matriptase is composed of an intracellular region in the amino terminus, followed by a transmembrane domain, one SEA, two CUB, four LDLR domains and a protease domain in the carboxyl terminus. HAI-1 has two Kunitz domains (KD1 and KD2) in the amino terminus, one LDLR domain between these two KDs, a transmembrane domain and a short intracellular region in the carboxyl terminus. Matriptase’s and HAI-1’s domain abbreviations: SEA, Sperm protein, Enterokinase and Agrin; CUB, C1r/C1s, Uegf and Bmp1; LDLR, LDL receptor¹⁰⁰. (D) Immunoblot analysis of total and activated matriptase as well as HAI-2 in CWR22Rv1, 103E and N2 cells. Forty micrograms of cell lysate per sample were separated by SDS-PAGE and immunoblotted using monoclonal M32, monoclonal M69 and polyclonal anti-HAI-2 antibodies to detect total matriptase (activated and latent matriptase), activated matriptase, and HAI-2 proteins, respectively. β-actin was used as a control.