PMC

Prostasin has important roles in chemoresistance and cell death in cell culture model. (a) Prostasin decreased in paclitaxel-resistance cancer cell line. O432: ovarian cancer cell line Ovca432 (sensitive to paclitaxel); O432-RP: paclitaxel resistance cell line generated from Ovca432. The prostasin protein levels in O432 and O432-RP cells are shown in immunoblots with specific antibodies. (b) Prostasin siRNAs transfection reduced prostasin. O432-pro-D cells (transfected with prostasin siRNA) express lower prostasin, compared with control cells of O432 (transfected with reagent only) and O432-Cs (transfected with no-targeting siRNA). The prostasin protein levels are shown in immunoblots with specific antibodies. (c) Downregulation of prostasin in O432 cells resulted in increase of chemoresistant activity. Cells were treated with paclitaxel at different concentrations for 24 h (starting 48 h after siRNAs transfection) and cultured with normal medium for an additional 7 to 10 days before cell survival was assayed. Relative cell survival rates of each cell line are shown. (d) Overexpression of prostasin greatly induces cell death in ovarian cancer cells. The cell survival rates are shown after forced overexpression of prostasin in several cell lines from day-0 to day-8, respectively. (e) Prostasin cDNA transfection resulted in overexpression of prostasin in chemoresistant O432-RP cells. O432-RP-pro-O cells (transfected with prostasin cDNA) express higher prostasin compared with control cells O432-RP and O432-RP-C (transfected with control vector). The prostasin protein levels are shown in immunoblots with specific antibodies. (f) Forced overexpression of prostasin represses growth of chemoresistant cells. Relative cell growth rates are shown for O432-RP-pro-O and control cells O432-RP and O432-RP-C. (g) Forced overexpression of prostasin in O432-RP cells re-sensitizes chemoresistant cells. Cells were plated at about 10–20% confluence and treated with paclitaxel at different concentrations for 24 h, cultured with normal medium for additional 7 to 10 days, then assayed for cell survival. Relative survival rates of cell lines are shown

Human foreskin lysates were prepared from 4 different donors in two batches with specimen 1–3 together and specimen 4 alone. Equal amount of protein from the four tissue lysates were analyzed by immunoblot for prostasin, matriptase, and HAI-1, as indicated (A and B). HAI-2 species were also analyzed in specimens 1–3 (C). The prostasin species include prostasin zymogen, active prostasin, and prostasin complex with HAI-1, as indicated. Matriptase species include matriptase zymogen and matriptase complex with HAI-1, as indicated. HAI-1 species include HAI-1 monomer, HAI-1 complexes with prostasin or matriptase, as indicated. HAI-2 species include the one with heavy N-glycan (C, right panel) and the one with light N-glycan (C, left panel).

A. The prostasin containing species present in Caco-2 cell lysates were separated and purified by sequential immunoaffinity chromatography using HAI-1 mAb M19-Sepharose, HAI-2 mAb DC-16 Sepharose, and then prostasin mAb YL11-Sepharose. The purified prostasin-HAI-1 complexes (lane 1), prostasin-HAI-2 complexes (lane 2), and prostasin monomers (lane 3) were analyzed for prostasin species by immunoblot using the prostasin-specific mAb YL11. B. Purified prostasin monomers (lane 1) were incubated with purified HAI-1 (lane 2) at 37°C for 5 min to allow the formation of prostasin-HAI-1 complexes (lane 3). The samples were then analyzed by western blot for prostasin-containing species. C. Purified prostasin monomers were incubated with a fluorogenic prostasin substrate and the kinetics of AMC release was recorded. Prostasin proteolytic activity assays were conducted at least four times. Representative data are shown. RFU stands for relative fluorescent unit.

Severe skin phenotype in transgenic mice with epidermal overexpression of wild type prostasin, zymogen-locked prostasin, and catalytically inactive prostasin. A–C, representative examples of the outward appearance of 12–15-day-old transgenic founders expressing wild type prostasin (A), zymogen-locked prostasin (B), and catalytically inactive prostasin (C) under the control of a keratin-5 promoter. A littermate without phenotype is shown on the left in A, and non-transgenic littermates are shown on the left for comparison in B and C. Note severe growth retardation, abnormal pelage hair, and skin ulcerations in transgenic founders expressing all three forms of prostasin. Shown are hematoxylin and eosin staining (D, G, J, and M), prostasin immunohistochemistry (E, H, K, and N), and Ki-67 immunohistochemistry (F, I, L, and O) of representative sections of dorsal skin from non-transgenic founders (D–F) and from founders expressing wild type (G–I), zymogen-locked (J–L), and catalytically inactive (M–O) prostasin under control of a keratin-5 promoter. The thickness of the epidermis is indicated by the vertical bar in D, G, J, and M, and the position of the basement membrane is indicated by the dotted line. Note the epidermal thickening and hyperkeratosis in wild type, zymogen-locked, and catalytically inactive prostasin transgenic founders, as compared with non-transgenic littermates. Insets in H, K, and N, high magnification images showing membrane-localized prostasin staining in keratinocytes. Examples of Ki-67-positive (proliferating) basal keratinocytes are shown with arrows in F, I, L, and O, and examples of proliferating suprabasal keratinocytes are shown with arrowheads in I, L, and O. Note the severe epidermal hyperproliferation in wild type prostasin, zymogen-locked prostasin, and catalytically inactive prostasin transgenic founders when compared with non-transgenic skin. Size bars, 50 μm (large panels) and 10 μm (insets).