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Active Plasma Kallikrein Localizes to Mast Cells and Regulates Epithelial Cell Apoptosis, Adipocyte Differentiation, and Stromal Remodeling during Mammary Gland Involution

Lilla, Jennifer N., Joshi, Ravi V., Craik, Charles S., Werb, Zena·2009·Journal of Biological Chemistry
DOI 10.1074/jbc.m900508200
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FIGURE 1 Ecotin-PKal inhibits mouse plasma kallikrein and binds PKal isolated from mammary gland lysates. A , binding assays were performed to determine Ki values for ecotin-PKal to mouse PKal or plasmin. After a 4-h incubation at 37 °C, remaining protease activity was measured using a p -nitroanilide substrate (see 'Experimental Procedures'). Reaction velocities were averaged over three trials and normalized to the baseline reaction velocity. The apparent Ki of ecotin-PKal for mouse PKal was determined to be 40nM;the Ki for mouseplasminwasdeterminedtobe60 𝛍 M. B , protein lysates from 4-day-involuting mammary glands were run over either streptavidinonly beads ( no ecotin-PKal ) or streptavidin beads bound with biotinylated ecotin-PKal ( + ecotin-PKal ). Flow-thru from the columns was collected, and bead-bound proteins were eluted by boiling the beads in sample buffer. All samples were subjected to electrophoresis and Western blotting using an antibody against mouse prekallikrein ( 𝛂 -mouse KLKB1, R&D Systems). The bands likely represent full-length PKal ( ∼ 80 kDa), its heavy chain (42 kDa, unglycosylated), and its light chain (28 kDa, unglycosylated). mPKal , mouse prekallikrein peptide, R&D Systems), against which the antibody was raised.
FIGURE 1 Ecotin-PKal inhibits mouse plasma kallikrein and binds PKal isolated from mammary gland lysates. A , binding assays were performed to determine Ki values for ecotin-PKal to mouse PKal or plasmin. After a 4-h incubation at 37 °C, remaining protease activity was measured using a p -nitroanilide substrate (see 'Experimental Procedures'). Reaction velocities were averaged over three trials and normalized to the baseline reaction velocity. The apparent Ki of ecotin-PKal for mouse PKal was determined to be 40nM;the Ki for mouseplasminwasdeterminedtobe60 𝛍 M. B , protein lysates from 4-day-involuting mammary glands were run over either streptavidinonly beads ( no ecotin-PKal ) or streptavidin beads bound with biotinylated ecotin-PKal ( + ecotin-PKal ). Flow-thru from the columns was collected, and bead-bound proteins were eluted by boiling the beads in sample buffer. All samples were subjected to electrophoresis and Western blotting using an antibody against mouse prekallikrein ( 𝛂 -mouse KLKB1, R&D Systems). The bands likely represent full-length PKal ( ∼ 80 kDa), its heavy chain (42 kDa, unglycosylated), and its light chain (28 kDa, unglycosylated). mPKal , mouse prekallikrein peptide, R&D Systems), against which the antibody was raised.CC-BY · From the PDF
Image 2
CC-BY · From the PDF
FIGURE 3 Biotinylated ecotin-PKal stains tissues with connective tissuetype mast cells but not liver. Frozen sections using 50 nM ecotin-PKal plus Alexa488-conjugatedstreptavidin,DAPIcounterstain. Scalebar for A-D is 100 𝛍 m. Active PKal is seen in mast cells of the skin ( A ) and tongue ( B ), but not in liver ( C ) or intestine ( D ). Active PKal cannot be seen in liver, where prekallikrein is synthesized but not activated, and therefore is not bound by ecotin-PKal. Active PKal is not seen in the small intestine, where mast cells are of the mucosal subtype.
FIGURE 3 Biotinylated ecotin-PKal stains tissues with connective tissuetype mast cells but not liver. Frozen sections using 50 nM ecotin-PKal plus Alexa488-conjugatedstreptavidin,DAPIcounterstain. Scalebar for A-D is 100 𝛍 m. Active PKal is seen in mast cells of the skin ( A ) and tongue ( B ), but not in liver ( C ) or intestine ( D ). Active PKal cannot be seen in liver, where prekallikrein is synthesized but not activated, and therefore is not bound by ecotin-PKal. Active PKal is not seen in the small intestine, where mast cells are of the mucosal subtype.CC-BY · From the PDF
Image 4
CC-BY · From the PDF
FIGURE5 Delayinmammaryglandinvolutionaftertreatmentwithecotin-PKal. Animals were treated twice daily during the first 4 days of involution with 100 𝛍 g of ecotin-PKal or with vehicle control. n = 8 mice/group. All error bars represent S.D. A , frozen sections of mammary glands stained with Oil Red O for lipid showecotin-PKal-treatedglandshavefewerandlessmatureadipocytes. Blackarrowheads indicate representative lipid staining. Scale bar is 1 mm. B , quantification of lipid contentbyOilRedOstainingshowsecotin-PKal-treatedglandshavesignificantlylesslipidafter4daysofinvolution.Student's t test: p = 0.041. C , paraffin sections of mammary glands stained with Masson's trichrome to detect collagen ( blue ) and cytoplasm ( pink ) show that ecotin-PKal-treated glands have more collagen depositionandmoreimmaturestromalcellsthancontrols. Blackarrowheads indicaterepresentativeareasofcollagendeposition. Scalebar is 100 𝛍 m. D , quantification ofcollagencontentbyMasson'strichromestainshowsecotin-PKal-treatedglandshavesignificantlymorecollagen.Student's t test: p = 0.003. All error bars represent S.D. E , paraffin sections of mammary glands stained with picro-sirius red to demonstrate by birefringence under polarized light the difference between thick ( orangeyellow ) and thin ( green ) collagen fibrils. Scale bar is 100 𝛍 m. F , abdominal mammary gland weights expressed as a ratio of overall body weight on day 5 of involution showthat ecotin-PKal-treated glands weigh less after treatment than controls. Student's t test: p = 0.029.
FIGURE5 Delayinmammaryglandinvolutionaftertreatmentwithecotin-PKal. Animals were treated twice daily during the first 4 days of involution with 100 𝛍 g of ecotin-PKal or with vehicle control. n = 8 mice/group. All error bars represent S.D. A , frozen sections of mammary glands stained with Oil Red O for lipid showecotin-PKal-treatedglandshavefewerandlessmatureadipocytes. Blackarrowheads indicate representative lipid staining. Scale bar is 1 mm. B , quantification of lipid contentbyOilRedOstainingshowsecotin-PKal-treatedglandshavesignificantlylesslipidafter4daysofinvolution.Student's t test: p = 0.041. C , paraffin sections of mammary glands stained with Masson's trichrome to detect collagen ( blue ) and cytoplasm ( pink ) show that ecotin-PKal-treated glands have more collagen depositionandmoreimmaturestromalcellsthancontrols. Blackarrowheads indicaterepresentativeareasofcollagendeposition. Scalebar is 100 𝛍 m. D , quantification ofcollagencontentbyMasson'strichromestainshowsecotin-PKal-treatedglandshavesignificantlymorecollagen.Student's t test: p = 0.003. All error bars represent S.D. E , paraffin sections of mammary glands stained with picro-sirius red to demonstrate by birefringence under polarized light the difference between thick ( orangeyellow ) and thin ( green ) collagen fibrils. Scale bar is 100 𝛍 m. F , abdominal mammary gland weights expressed as a ratio of overall body weight on day 5 of involution showthat ecotin-PKal-treated glands weigh less after treatment than controls. Student's t test: p = 0.029.CC-BY · From the PDF
Image 6
CC-BY · From the PDF
FIGURE 7 Ecotin-PKal-treated mammary glands have an altered epithelial-stromal ratio during treatment. Animals were treated as described, n = 2 mice/ group/timepoint. A , frozen sections of mammaryglandsstainedwithOilRedOforlipidshowecotin-PKal-treatedglandshavefewerandlessmatureadipocytesinthe second phase of involution, even 5 days after end of treatment. Scale bar is 100 𝛍 m. B , ecotin-PKal-treated mammary glands weigh more during the first apoptotic phaseofinvolution,thenweighlessduringthestromalremodelingphase.Abdominalmammaryglandweightsareexpressedasaratioofoverallbodyweightondays 1-5andday9ofinvolution.Ecotin-PKaltreatmentoccurredduringdays1-5. C , quantification of lipid content by Oil Red O staining shows ecotin-PKal-treated glands have less lipid even after 5 days of recovery from ecotin-PKal treatment. Shaded area marks ecotin-PKal treatment. Error bars represent S.D.
FIGURE 7 Ecotin-PKal-treated mammary glands have an altered epithelial-stromal ratio during treatment. Animals were treated as described, n = 2 mice/ group/timepoint. A , frozen sections of mammaryglandsstainedwithOilRedOforlipidshowecotin-PKal-treatedglandshavefewerandlessmatureadipocytesinthe second phase of involution, even 5 days after end of treatment. Scale bar is 100 𝛍 m. B , ecotin-PKal-treated mammary glands weigh more during the first apoptotic phaseofinvolution,thenweighlessduringthestromalremodelingphase.Abdominalmammaryglandweightsareexpressedasaratioofoverallbodyweightondays 1-5andday9ofinvolution.Ecotin-PKaltreatmentoccurredduringdays1-5. C , quantification of lipid content by Oil Red O staining shows ecotin-PKal-treated glands have less lipid even after 5 days of recovery from ecotin-PKal treatment. Shaded area marks ecotin-PKal treatment. Error bars represent S.D.CC-BY · From the PDF
FIGURE8 Modelofthefunctionofplasmakallikreinduringmammarygland involution. As loss of tPA and uPA (in gray ) does not recapitulate the effect of plasminogendeficiencyinmammaryglandinvolution,wepositthatplasmakallikrein (in bold ) is the critical activator of plasminogen during this process. Active PKal is required for apoptosis of the secretory epithelium, subsequent stromal remodeling, and eventual adipocyte replenishment of the mammary gland.
FIGURE8 Modelofthefunctionofplasmakallikreinduringmammarygland involution. As loss of tPA and uPA (in gray ) does not recapitulate the effect of plasminogendeficiencyinmammaryglandinvolution,wepositthatplasmakallikrein (in bold ) is the critical activator of plasminogen during this process. Active PKal is required for apoptosis of the secretory epithelium, subsequent stromal remodeling, and eventual adipocyte replenishment of the mammary gland.CC-BY · From the PDF

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Authors
Lilla, J. N., Joshi, R. V., Craik, C. S., & Werb, Z.
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2009-05
Volume
284
Issue
20
Pages
13792-13803
ISSN
0021-9258
DOI
10.1074/jbc.m900508200
Cited by
50
References
0
Language
en
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