1. Plasminogen-Dependent Matriptase Activation Accelerates Plasmin Generation by Differentiating Primary Human Keratinocytes 
Ya-Wen Chen, Shi Yin, Ying-Jung J. Lai, Michael D. Johnson, Chen-Yong Lin 
Journal of Investigative Dermatology 
Volume 136, Issue 6, Pages (June 2016)
DOI: /j.jid
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2. Figure 1
Differentiating human keratinocytes activate matriptase in response to serum. Primary human keratinocytes were grown in low (0.09 mM) calcium basal media (control, lane C), supplemented with calcium (Ca) and/or either FBS or human serum (HS). (a, b) The concentrations of calcium and FBS were as indicated and the cells were cultured for 16 hours. (c–e) HS was at 2% and the concentration of calcium was 0.2 mM. (c) The cells were treated with HS for 16 hours, followed by 0.2 mM calcium for another 16 hours (lane HS → Ca) or in reverse order (lane Ca → HS). (d) The cells were primed with 0.2 mM calcium for 16 hours, followed by HS treatment for the indicated times. (e) The cells were primed with 0.2 mM calcium for the indicated times, followed by HS treatment for 2 hours. The cells were analyzed for the presence of different matriptase species by immunoblot using the total matriptase mAb M24. Matriptase zymogen was detected as a 70-kDa protein band, and the appearance of the 120-kDa activated matriptase-HAI-1 complex indicated the zymogen activation of matriptase. Activated matriptase in complexes with HAI-1 was indicated by an arrow. Each experiment was repeated at least three times, n ≥ 3. FBS, fetal bovine serum; HAI-1, hepatocyte growth factor activator inhibitor-1; mAb, monoclonal antibody.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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3. Figure 2
De novo protein synthesis and intracellular signaling are required for serum-induced matriptase zymogen activation in differentiating keratinocytes. Primary human keratinocytes were maintained for 16 hours in medium, supplemented with 0.2 mM calcium and 2% human serum (lanes 0). The cells were treated with increasing amounts of (a) the mRNA synthesis inhibitor actinomycin D, (b) the protein synthesis inhibitor cycloheximide, (c) the intracellular calcium chelator BAPTA-AM, (d) the PKC inhibitor Ro , and (e) the PI3K inhibitor Ly The cells were lysed and analyzed for matriptase zymogen activation by immunoblot using the total matriptase mAb M24. Arrows indicate activated matriptase complexes with HAI-1. Each experiment was repeated at least three times, n ≥ 3. HAI-1, hepatocyte growth factor activator inhibitor-1; mAb, monoclonal antibody; PI3K, phosphatidylinositol 3-kinases; PKC, protein kinase C.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

4. Figure 3
Plasminogen is the serum factor that induces matriptase activation in differentiating keratinocytes. (a) Primary human keratinocytes were grown in low (0.09 mM) calcium basal media (control, lane 1), supplemented with calcium (Ca, lanes 2–5) and FBS (lane 3) or charcoal-stripped FBS (lane 4), or boiled FBS (lane 5). The cell lysates were analyzed for matriptase activation by western blot using matriptase mAb M24. (b) The purified serum factor that induces matriptase zymogen activation was mixed with SDS sample buffer in the absence (lanes 1 and 2) or presence (lane 3) of dithiothreitol and incubated at room temperature (lane 1) or 95 °C (lanes 2 and 3) for 5 minutes. The samples were analyzed by SDS-PAGE and visualized by staining the gel with ProtoBlue Safe. (c) The serum factor was subjected to in-gel trypsinization and mass spectrometry-proteomic analysis-based protein identification. The obtained sequences were matched to bovine plasminogen and are presented in single letter code with the amino acid sequence number of bovine plasminogen. (d) Primary human keratinocytes were stimulated with increasing amounts of plasminogen, as indicated, in the absence or presence of 0.2 mM calcium. The cells were analyzed for matriptase zymogen activation by immunoblot using mAb M24. (e) Primary human keratinocytes were maintained in 0.09 mM calcium (lanes 1–7) or 0.2 mM calcium (lanes 8–14). These cells were then treated with 1 μg/ml plasmin for 5 minutes (lanes 2 and 9), 10 minutes (lanes 3 and 10), 30 minutes (lanes 4 and 11), 60 minutes (lanes 5 and 12), 90 minutes (lanes 6 and 13), and 20 hours (lanes 7 and 14). Cell lysates were subjected to immunoblot analyses using a total matriptase mAb, M24. (f) Primary human keratinocytes were grown in the low calcium basal medium alone (lane 1), or supplemented with 0.2 mM calcium (lane 2), 0.1 M 6-AHA (lane 3), a combination of 0.2 mM calcium and 0.1 M 6-AHA (lane 4), a combination of 0.1 M 6-AHA and 1 μg/ml plasminogen (lane 5), a combination of 0.2 mM calcium and 1 μg/ml plasminogen (lane 6), or a combination of 0.2 mM calcium, 0.1 M 6-AHA, and 1 μg/ml plasminogen (lane 7). The cells were lysed and analyzed for matriptase zymogen activation by immunoblot using the matriptase mAb, M24. Activated matriptase in complexes with HAI-1 is indicated by an arrow. Each experiment was repeated at least three times, n ≥ 3, except the mass spectrometry-proteomic analysis (c). 6-AHA, 6-aminohexanoic acid; FBS, fetal bovine serum; HAI-1, hepatocyte growth factor activator inhibitor-1; mAb, monoclonal antibody.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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5. Figure 4
Plasmin generation is accelerated by matriptase zymogen activation. (a) Primary human keratinocytes were incubated with 50 nM plasminogen in the absence (C+PLG) or presence of 0.2 mM calcium (Ca+PLG). Generation of plasmin was monitored using the synthetic fluorogenic substrate, Boc-Val-Leu-Lys-AMC. For the insert, the rates of cleavage of the substrate in the presence of calcium were compared with the rate in the absence of calcium as a percentage at 48 minutes. (b) Primary human keratinocytes were preincubated with rat IgG, as a control, or various amounts of the rat-derived matriptase mAb 21–9 for 2 hours, followed by raising the calcium concentration to 0.2 mM and the addition of 50 nM plasminogen. Plasmin generation was assayed using the synthetic fluorogenic substrate, Boc-Val-Leu-Lys-AMC. For the insert, the rates of cleavage of the substrate in the presence matriptase mAb 21-9 were compared with the rate of the control using rat IgG as a percentage at 48 minutes. (c) Primary human keratinocytes were preincubated with DMSO, as a control, or either of the two small molecule matriptase zymogen activation inhibitors, F or F for 2 hours, followed by raising the calcium concentration to 0.2 mM and the addition of 50 nM plasminogen. Plasmin activity was monitored by the cleavage of the synthetic fluorogenic substrate, Boc-Val-Leu-Lys-AMC. For the insert, the rates of cleavage of the substrate in the presence of matriptase zymogen activation inhibitors were compared with the rate of the control with DMSO as a percentage at 48 minutes. All data presented are representative of three independent experiments performed in triplicate. Each experiment was repeated at least three times, n ≥ 3. AMC, 7-amino-4-methylcoumarin; DMSO, dimethyl sulfoxide; IgG, immunoglobulin G; mAb, monoclonal antibody.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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6. Figure 5
Pericellular active matriptase accelerates plasmin generation at the cell surface. (a, b) Primary human keratinocytes were stimulated to activate matriptase by a pH 6.0 buffer treatment (lane 2) or Dulbecco's phosphate-buffered saline as the nonactivation control (lane 1). The amount of zymogen activation was analyzed by immunoblot for the formation of the 120-kDa activated matriptase-HAI-1 complex at the cost of the 70-kDa matriptase zymogen in cell lysates (a, comparing lane 2 with lane 1) or by the cleavage of a synthetic fluorogenic substrate Boc-Gln-Ala-Arg-AMC by the shed active matriptase (b). (c) Primary human keratinocytes were induced to activate matriptase with a pH 6.0 buffer treatment. The cells (cellular), the conditioned buffer (Shed), or the combination of the cells and conditioned buffer (Combined) were analyzed for their ability to generate plasmin by the cleavage of the synthetic fluorogenic substrate, Boc-Val-Leu-Lys-AMC. For insert, the rates of cleavage of the substrate for the conditioned buffer alone and the cells alone were compared with the rate for the combination at 48 minutes as a percentage. (d) Primary human keratinocytes were incubated with plasminogen for the generation of plasmin in the presence of increasing amounts of 6-AHA. The generation of plasmin was monitored by the cleavage of the synthetic fluorogenic substrate, Boc-Val-Leu-Lys-AMC. For insert, the rates of cleavage of the substrate in the presence of 6-AHA were compared with the rate of the control in the absence of 6-AHA at 48 minutes as a percentage. All data presented are representative of three independent experiments performed in triplicate. Each experiment was repeated at least three times, n ≥ 3. 6-AHA, 6-aminohexanoic acid; AMC, 7-amino-4-methylcoumarin; HAI-1, hepatocyte growth factor activator inhibitor-1.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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7. Figure 6
A schematic model for a putative feed-forward mechanism for matriptase zymogen and plasmin generation. A feed-forward mechanism by which differentiating keratinocytes accelerate plasmin generation via plasminogen-induced matriptase zymogen activation is proposed. Calcium-primed differentiating human keratinocytes activate matriptase in response to exposure to extracellular plasminogen. The resultant pericellular active matriptase then returns to the cell surface to activate plasminogen activator, leading to the acceleration of plasmin generation.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions