Figure 1.
Shedding of active matriptase by human keratinocyte.
A. Human keratinocyte HaCaT cells were incubated either with PBS (C) as a control or a pH 6.0 buffer (Acid, A) to induce activation of matriptase. Cell lysates were analyzed by immunoblot for matriptase (MTP) or HAI-1 (HAI-1). B. HaCaT cells were incubated with a pH 6.0 buffer to induce matriptase activation. The cells and the conditioned buffer together (Combined), the cells alone (Cellular) and the conditioned buffer alone (Shed) were analyzed for matriptase activity using a matriptase synthetic fluorescent substrate, Boc-Gln-Ala-Arg-AMC. Data are representative of four independent experiments done under similar conditions. C. The conditioned buffer was subjected to immunoprecipitation with the activated matriptase mAb M69. The loading control (Loading), the unbound fraction (Unbound), and the eluent (Elution) were assayed for matriptase activity using the substrate, Boc-Gln-Ala-Arg-AMC. Data are representative of three independent experiments done under similar conditions. D. HaCaT cells were incubated with DPBS as control or a pH 6.0 buffer to induce matriptase activation. The conditioned buffer was collected and subjected to gelatin zymography.
Figure 2.
Activation of pro-HGF and acceleration of plasminogen activation by active matriptase shed from human keratinocytes.
a. Pro-HGF was incubated with increasing amounts of active matriptase, as indicated, either with or without HaCaT cells at 37°C for 30 min. Samples were analyzed by immunoblot for HGF cleavage using an antibody directed against the beta subunit of HGF. b. Primary human keratinocytes were incubated with DPBS (No Activation) or a pH 6.0 buffer (Activation) to induce activation of matriptase, after which the buffer on the cells was adjusted to pH 7.5. Plasminogen (50 nM) was then added to the cells and the generation of plasmin was monitored by the cleavage of a plasmin synthetic fluorescent substrate, Boc-Val-Leu-Lys-AMC. Data are representative of three independent experiments done under similar conditions. c. Primary human keratinocytes were incubated with a pH 6.0 buffer to induce matriptase activation, followed by buffering to pH 7.5. Plasminogen (50 nM) was then added to the cells in the presence of the shed fractions (combined) or in the absence of the shed fractions (Cellular) or the shed fraction alone (Shed). Generation of plasmin was monitored by the cleavage of Boc-Val-Leu-Lys-AMC. Data are representative of four independent experiments done under similar conditions.
Figure 3.
Identification of AT as a component of the novel 110-kDa matriptase complex.
a. Human keratinocytes (HaCaT), mammary (184 A1N4) and prostate (RWPE1) epithelial cells were incubated with a pH 6.0 buffer to induce matriptase activation. The conditioned buffers (S) and the total cell lysates (T) were analyzed by immunoblot for total matriptase. Arrow indicates the 110-kDa matriptase complex. b. HaCaT cells were incubated with a pH 6.0 buffer for the indicated times (minutes). The cell lysates and the conditioned buffers were subjected to immunoblot analyses for total matriptase. c. Left panel: Purified 110-kDa matriptase complex was analyzed by SDS-PAGE under non-reducing and boiled conditions (NR, B). The protein bands were visualized by staining with ProtoBlue Safe. The protein bands a and b, as indicated, were subjected to protein identification by MS/MS (see Fig. S2 for sequence data). Middle and right panels: HaCaT cells, pretreated with human serum, were induced to activate matriptase by pH 6.0 treatment. Cell lysates and the concentrated conditioned buffers were subjected to immunoblot analyses by immunoblot for total matriptase (Total MTP) and AT (AT).
Figure 4.
The shedding of active matriptase is inversely correlated with the levels of membrane-bound AT.
A. HaCaT cells were incubated with the three AT preparations, as indicated, at 37°C for 5 min. The cellular retention of AT were analyzed by immunoblot analyses using an AT antibody. B. The AT-pretreated cells were then induced to activate matriptase by a pH 6.0 buffer. The cell lysates and the conditioned buffers were analyzed for matriptase species and AT species, as indicated. C. Matriptase activity in the conditioned buffer was also assessed by the cleavage of Boc-Gln-Ala-Arg-AMC. Data was done in triplicate and is a representative example of three independent experiments.
Figure 5.
Syndecan-1 can be robustly shed by active matriptase.
HaCaT cells were incubated with increasing active matriptase, as indicated, at 37°C for 30 min. The conditioned media were collected and subjected to dot blot analysis with two different loaded volumes (1× and 2×) for syndecan-1. Data are representative of three independent experiments done under similar conditions.
Figure 6.
A schematic model of the different functional and regulatory mechanisms for matriptase control and activity that relate to the histological differences between simple/polarized and stratified epithelium.
The subcellular distribution of matriptase (MTP), prostasin, and HAI-1 and the events following matriptase activation in the simple/polarized and the stratified epithelium are presented schematically. In simple/polarized epithelium, matriptase is targeted to the basolateral surface and prostasin is targeted to the apical surface with HAI-1 targeted to the both cell surface subdomains. The polarized expression of matriptase and prostasin is, however, likely not present in stratified epithelium, and as a result, matriptase gains an access to prostasin. When high level matriptase activation is induced, active matriptase is rapidly inhibited by nearby HAI-1 and the short-lived active matriptase can act on prostasin only in keratinocytes and not in polarized epithelial cells, due to the differential subcellular distributions of matriptase and prostasin between the two epithelia. Active prostasin is also rapidly inhibited by HAI-1 by forming a complex. A proportion of the active matriptase is shed from cell surface, an event that is evident in the stratified epithelial cells, and not in polarized epithelial cells. The shedding of active matriptase, the inhibition of active matriptase by AT bound to the cell via surface heparan sulfate proteoglycans, such as syndecans, is more obvious and important in stratified epithelial cells than in polarized epithelial cells. The active matriptase that escapes inactivation by HAI-1 or AT, can then act on its substrates, such as PAR-2, pro-uPA, pro-HGF, and syndecans.