Active Plasma Kallikrein Localizes to Mast Cells and Regulates Epithelial Cell Apoptosis, Adipocyte Differentiation, and Stromal Remodeling during Mammary Gland Involution
2009; Elsevier BV; Volume: 284; Issue: 20 Linguagem: Inglês
10.1074/jbc.m900508200
ISSN1083-351X
AutoresJennifer N. Lilla, Ravi V. Joshi, Charles S. Craik, Zena Werb,
Tópico(s)Estrogen and related hormone effects
ResumoThe plasminogen cascade of serine proteases directs both development and tumorigenesis in the mammary gland. Plasminogen can be activated to plasmin by urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), and plasma kallikrein (PKal). The dominant plasminogen activator for mammary involution is PKal, a serine protease that participates in the contact activation system of blood coagulation. We observed that the prekallikrein gene (Klkb1) is expressed highly in the mammary gland during stromal remodeling periods including puberty and postlactational involution. We used a variant of ecotin (ecotin-PKal), a macromolecular inhibitor of serine proteases engineered to be highly specific for active PKal, to demonstrate that inhibition of PKal with ecotin-PKal delays alveolar apoptosis, adipocyte replenishment, and stromal remodeling in the involuting mammary gland, producing a phenotype resembling that resulting from plasminogen deficiency. Using biotinylated ecotin-PKal, we localized active PKal to connective tissue-type mast cells in the mammary gland. Taken together, these results implicate PKal as an effector of the plasminogen cascade during mammary development. The plasminogen cascade of serine proteases directs both development and tumorigenesis in the mammary gland. Plasminogen can be activated to plasmin by urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), and plasma kallikrein (PKal). The dominant plasminogen activator for mammary involution is PKal, a serine protease that participates in the contact activation system of blood coagulation. We observed that the prekallikrein gene (Klkb1) is expressed highly in the mammary gland during stromal remodeling periods including puberty and postlactational involution. We used a variant of ecotin (ecotin-PKal), a macromolecular inhibitor of serine proteases engineered to be highly specific for active PKal, to demonstrate that inhibition of PKal with ecotin-PKal delays alveolar apoptosis, adipocyte replenishment, and stromal remodeling in the involuting mammary gland, producing a phenotype resembling that resulting from plasminogen deficiency. Using biotinylated ecotin-PKal, we localized active PKal to connective tissue-type mast cells in the mammary gland. Taken together, these results implicate PKal as an effector of the plasminogen cascade during mammary development. The plasminogen cascade of serine proteases regulates both development and tumorigenesis in the mammary gland (1Dano K. Behrendt N. Hoyer-Hansen G. Johnsen M. Lund L.R. Ploug M. Romer J. Thromb. Haemost. 2005; 93: 676-681Crossref PubMed Scopus (385) Google Scholar, 2Lund L.R. Bjorn S.F. Sternlicht M.D. Nielsen B.S. Solberg H. Usher P.A. Osterby R. Christensen I.J. Stephens R.W. Bugge T.H. Dano K. Werb Z. Development. 2000; 127: 4481-4492PubMed Google Scholar). The ultimate effector in this cascade, plasminogen as its active form, plasmin, is mediated by an intricate cascade of plasminogen activators and protease inhibitors. Plasminogen-deficient mice exhibit significant defects in lactational competence and post-lactational mammary gland involution (2Lund L.R. Bjorn S.F. Sternlicht M.D. Nielsen B.S. Solberg H. Usher P.A. Osterby R. Christensen I.J. Stephens R.W. Bugge T.H. Dano K. Werb Z. Development. 2000; 127: 4481-4492PubMed Google Scholar), the process by which the differentiated, lactating gland remodels after the cessation of lactation to a state approaching that of the non-pregnant animal. The effect of plasminogen loss is exacerbated after a round of pregnancy and lactation: plasminogen-null mammary glands have poorly developed secretory alveoli during lactation, and upon involution, never fully involute. Instead, the secretory alveoli fail to regress normally. Moreover, the stroma becomes fibrotic and is cleared incompletely of partially degraded epithelial basement membrane. Because plasminogen-deficient mice largely are unable to support a second round of pregnancy and lactation (2Lund L.R. Bjorn S.F. Sternlicht M.D. Nielsen B.S. Solberg H. Usher P.A. Osterby R. Christensen I.J. Stephens R.W. Bugge T.H. Dano K. Werb Z. Development. 2000; 127: 4481-4492PubMed Google Scholar), this suggests that the involution defect is not overcome by activities of other proteases eventually. These studies establish plasminogen as a crucial protease in normal mammary gland biology.Plasminogen is synthesized in the liver and circulates as a zymogen through blood plasma to all vascularized tissues of the body. As this expression and circulation are constant, activation of the plasminogen cascade must be controlled locally to avoid rampant tissue proteolysis. Accordingly, plasminogen can be activated to plasmin by urokinase-type plasminogen activator (uPA), 2The abbreviations used are: uPA, urokinase-type plasminogen activator; tPA, tissue-type plasminogen activator; PKal, plasma kallikrein; DPPI, dipeptidyl peptidase I; ECL, enhanced chemiluminescence; DAB, 3,3′-diaminobenzidine; RT-qPCR, real-time quantitative polymerase chain reaction; HPRT, hypoxanthine phosphoribosyltransferase; RQ, quantile regression; ECM, extracellular matrix; CAE, chloroacetate esterase; PBS, phosphate-buffered saline; DAPI, 4′,6-diamidino-2-phenylindole. 2The abbreviations used are: uPA, urokinase-type plasminogen activator; tPA, tissue-type plasminogen activator; PKal, plasma kallikrein; DPPI, dipeptidyl peptidase I; ECL, enhanced chemiluminescence; DAB, 3,3′-diaminobenzidine; RT-qPCR, real-time quantitative polymerase chain reaction; HPRT, hypoxanthine phosphoribosyltransferase; RQ, quantile regression; ECM, extracellular matrix; CAE, chloroacetate esterase; PBS, phosphate-buffered saline; DAPI, 4′,6-diamidino-2-phenylindole. tissue-type plasminogen activator (tPA), and plasma kallikrein (3Miles L.A. Greengard J.S. Griffin J.H. Thromb. Res. 1983; 29: 407-417Abstract Full Text PDF PubMed Scopus (75) Google Scholar). Though tPA and uPA are efficient and well characterized plasminogen activators, studies of mice singly as well as doubly targeted for deficiency of these plasminogen activators show they do not recapitulate the mammary gland phenotype of plasminogen deficiency (4Selvarajan S. Lund L.R. Takeuchi T. Craik C.S. Werb Z. Nat. Cell Biol. 2001; 3: 267-275Crossref PubMed Scopus (128) Google Scholar). Instead, through use of variants of ecotin, a macromolecular inhibitor for serine proteases derived from Escherichia coli, we have previously suggested that the dominant plasminogen activator for mammary stromal involution is plasma kallikrein (PKal) (4Selvarajan S. Lund L.R. Takeuchi T. Craik C.S. Werb Z. Nat. Cell Biol. 2001; 3: 267-275Crossref PubMed Scopus (128) Google Scholar).PKal, the activated form of the zymogen prekallikrein encoded by the Klkb1 gene, is an 80-kDa serine protease that also is synthesized in the liver and circulates in plasma at about 40-50 μg/ml. PKal participates in the contact activation system of intrinsic coagulation by activating high molecular weight kininogen into bradykinin (5Baumgarten C.R. Nichols R.C. Naclerio R.M. Lichtenstein L.M. Norman P.S. Proud D. J. Immunol. 1986; 137: 977-982PubMed Google Scholar, 6Motta G. Rojkjaer R. Hasan A.A. Cines D.B. Schmaier A.H. Blood. 1998; 91: 516-528Crossref PubMed Google Scholar, 7Rojkjaer R. Schmaier A.H. Immunopharmacology. 1999; 43: 109-114Crossref PubMed Scopus (29) Google Scholar, 8Schmaier A.H. Rojkjaer R. Shariat-Madar Z. Thromb. Haemost. 1999; 82: 226-233Crossref PubMed Scopus (43) Google Scholar). While plasma kallikrein is so-named due to its bradykinin-generating ability, it is in fact structurally and catalytically distinct from the large family of tissue kallikreins, which activate an alternate form of bradykinin from both high and low molecular weight kininogen (9Lima A.R. Alves F.M. Angelo P.F. Andrade D. Blaber S.I. Blaber M. Juliano L. Juliano M.A. Biol. Chem. 2008; 389: 1487-1494Crossref PubMed Scopus (9) Google Scholar). Moreover, PKal activates plasminogen into plasmin in vitro (3Miles L.A. Greengard J.S. Griffin J.H. Thromb. Res. 1983; 29: 407-417Abstract Full Text PDF PubMed Scopus (75) Google Scholar), albeit less efficiently than uPA and tPA.To determine the role of PKal in plasminogen activation in vivo in mammary gland involution, we used a variant of ecotin that was engineered to be highly specific for active PKal (10Stoop A.A. Craik C.S. Nat. Biotechnol. 2003; 21: 1063-1068Crossref PubMed Scopus (55) Google Scholar). This ecotin variant, named ecotin-PKal, inhibits plasminogen activation in vivo in a model of wound healing (11Lund L.R. Green K.A. Stoop A.A. Ploug M. Almholt K. Lilla J. Nielsen B.S. Christensen I.J. Craik C.S. Werb Z. Dano K. Romer J. EMBO J. 2006; 25: 2686-2697Crossref PubMed Scopus (114) Google Scholar). In this study, we demonstrate that inhibition of PKal significantly delays mammary gland involution.EXPERIMENTAL PROCEDURESExperimental Animals—Care of animals and all animal experiments were performed in accordance with protocols approved by the UCSF Institutional Animal Use and Care Committee (IACUC). For postlactational mammary gland involution studies, pregnant female CD1 mice (Charles River Laboratories) gave birth naturally, then offspring number was normalized to 8 per mouse for each experimental animal. Offspring were weaned after 10 days of lactation, which became day 0 of involution. On days 1-4 of involution, mice were injected intraperitoneally every 12 h with either normal saline or 100 μg of ecotin-PKal as previously described (4Selvarajan S. Lund L.R. Takeuchi T. Craik C.S. Werb Z. Nat. Cell Biol. 2001; 3: 267-275Crossref PubMed Scopus (128) Google Scholar). Animals were sacrificed on day 5 of involution (n = 8/group) and mammary glands were harvested. Animals were also sacrificed on each of days 1-4 of involution or were treated for days 1-4 then allowed to recover until sacrifice on day 9 of involution (n = 2/time point).For PKal localization experiments, wild-type FVB and CD1 mice were obtained from Charles River Laboratories. C57BL/6J-KitW-sh/W-sh mice were obtained from the Jackson Laboratory and C57BL/6 mice were obtained from Charles River Laboratories. Dipeptidyl peptidase I (DPPI (-/-)) knock-out mice (12Pham C.T. Ley T.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8627-8632Crossref PubMed Scopus (343) Google Scholar) were provided by Dr. Lisa Coussens (UCSF). For all mouse mammary models, thoracic mammary glands 2 and 3 were removed for RNA or protein collection, and/or abdominal mammary glands 4 were removed for microscopic analysis. To inhibit mast cell degranulation, CD1 female mice (Charles River Laboratories) were injected intraperitoneally with 50 mg/kg body weight sodium cromoglycate (Sigma) dissolved in saline as previously described (13Jamieson T. Cook D.N. Nibbs R.J. Rot A. Nixon C. McLean P. Alcami A. Lira S.A. Wiekowski M. Graham G.J. Nat. Immunol. 2005; 6: 403-411Crossref PubMed Scopus (251) Google Scholar), and then 2 h later, mammary glands were removed for analysis.Preparation of Ecotin-PKal—A pTacTac expression vector containing the Ecotin-PKal gene was transformed into XL-1 blue (Stratagene, La Jolla, CA) competent E. coli and grown at 37 °C in 2xYT media with carbenicillin at 100 μg/ml to an A600 = 0.4. Isopropyl-β-d-thiogalactoside was added to 0.5 mm concentration and incubated overnight at 37 °C. The cell culture was harvested by centrifugation, and the cell pellet was resuspended in 25% sucrose 10 mm Tris-HCl, pH 8.0. The cell suspension was then treated for 2 h with lysozyme and EDTA at final concentrations of 1.5 mg/ml and 50 mm, respectively, to release the periplasmic protein content. The cell debris was pelleted, and the resulting supernatant was dialyzed against 1 mm HCl. The dialysate was again centrifuged to remove any precipitated material and then adjusted to pH > 7.0 with 1 m Tris, pH 8.0. NaCl was added to the supernatant to a final concentration of 300 mm and the solution was boiled in water at 100 °C for 10 min. Denatured material was removed by centrifugation, and the solution was dialyzed against distilled water overnight. The dialysate was concentrated to <5 ml by membrane ultrafiltration using Amicon YM-10, (Millipore, Billerica, MA) and then syringe-filtered (0.22 μm). Trifluoroacetic acid was added to 0.1% and Ecotin-PKal was purified from a C4 semi-prep column (Vydac, Deerfield, IL) using an acetonitrile gradient. Ecotin-PKal typically eluted at 35% acetonitrile, 0.1% trifluoroacetic acid water (v/v). Fractions corresponding to Ecotin-PKal were lyophilized, re-dissolved in distilled water, and stored at 4 °C for later use.Ecotin-PKal Inhibition Kinetics—Active mouse plasmin was obtained from Haematologic Technologies Inc. (Essex Junction, VT). Recombinant mouse plasma kallikrein (R&D Systems, Minneapolis, MN) was obtained as a pro-enzyme and activated with thermolysin (R&D Systems) as described by the manufacturer. All binding titrations and dilutions of reagents were carried out in reaction buffer: 50 mm Tris pH 7.5, 150 mm NaCl, 0.005% Tween 20. Ecotin-PKal was diluted to final concentrations ranging from 1 nm to 100 μm for plasmin assays and from 0.1 pm to 10 μm for plasma kallikrein assays. In the binding assays, plasmin and plasma kallikrein were present at 10 nm and 1 nm, respectively. The reactions were performed in triplicate in a 96-well plate with a final volume of 250 μl. The reactants were incubated at 37 °C for 4 h. After the incubation time, the activity was measured by addition of a p-nitroanilide peptide substrate and then by monitoring the absorbance (λ = 405 nm) at room temperature. Plasmin activity was measured using the S-2251 substrate (Chromogenix, Milano, Italy) at a final concentration of 600 μm. Plasma kallikrein activity was measured using the S-2232 substrate (Chromogenix) at a final concentration of 800 μm. Initial velocities of the reaction were calculated using Softmax Pro Software (Molecular Devices, Sunnyvale, CA). The rates were averaged over all three trials. The average rates were normalized to the rate of the negative control (0 nm ecotin-PKal) sample to yield V/Vmax. The rates were plotted versus ligand (ecotin-PKal) concentration and fitted to V/Vmax = 1 - L/(IC50 + L) using Kaleidograph (Synergy Software, Reading, PA) to determine the Ki for each of the enzymes.Western Blotting and Affinity Chromatography—Mammary glands were lysed in pH 8 radioimmune precipitation assay buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, and 0.1% sodium dodecyl sulfate (SDS)) plus complete protease inhibitors, homogenized, then centrifuged at 14,000 rpm at 4 °C to collect supernatant. Ecotin-PKal biotinylated with Sulfo-NHS-LC-LC biotin (EZ-Link, Pierce) was used to generate affinity chromatography columns using immobilized streptavidin beads (Pierce). Mammary lysates (1 mg) were added to the columns and incubated for 2 h at 4 °C. PKal was detected by Western blotting of mammary lysates, column flow-thru, and column beads boiled in sample buffer. Samples were separated by electrophoresis under reducing, denaturing conditions on 4-12% bis-Tris acrylamide gels (Invitrogen, Carlsbad, CA) and then blotted onto polyvinylidene difluoride membranes (Amersham Biosciences, Piscat-away, NJ). Membranes were blocked with 5% nonfat milk and 0.1% Tween in PBS and incubated overnight with a polyclonal antibody raised against recombinant mouse KLKB1 (1:1000, R&D Systems) in blocking solution. Membranes were washed and incubated with donkey anti-goat IgG (1:2000, Amersham Biosciences) before detection with ECL chemiluminescent reagent (Amersham Biosciences).Histology, Immunohistochemistry, and Immunofluorescence—Right no. 4 mammary glands were removed, weighed, and immediately embedded in OCT (Sakura, Torrance, CA) medium on dry ice. Left no. 4 mammary glands were removed and fixed overnight in 4% paraformaldehyde at 4 °C, then processed for paraffin embedding. 5-μm frozen or paraffin sections were cut for use in histology, enzyme histochemistry, immunohistochemistry, and immunofluorescence. To visualize gland lipid content, frozen sections were fixed additionally overnight in 4% paraformaldehyde, then rinsed in PBS, and air-dried for ≥3 h. Slides were then stained in Oil Red O (60% saturated Oil Red O (Sigma, 0.5% in isopropyl alcohol); 40% (1% type III corn dextrin (Sigma) in distilled water)) for 20 min. Slides were rinsed in PBS then counterstained briefly in Gill's hematoxylin (Sigma) and rinsed and stored in PBS. To examine overall gland morphology and stromal composition, Masson's Trichrome (Sigma) was used to stain paraffin sections according to the manufacturer's instructions. To visualize collagen content in the glands, paraffin sections were stained with 0.1% sirius red in saturated picric acid (picro-sirius red) (14Junqueira L.C. Bignolas G. Brentani R.R. Histochem. J. 1979; 11: 447-455Crossref PubMed Scopus (1943) Google Scholar) and observed under polarizing light. We identified apoptotic cells in paraffin sections of mammary tissue using an antibody against caspase-cleaved cytokeratin 18 (M30 CytoDEATH, Roche, Mannheim, Germany), a marker of cells undergoing apoptosis (15Leers M.P. Kolgen W. Bjorklund V. Bergman T. Tribbick G. Persson B. Bjorklund P. Ramaekers F.C. Bjorklund B. Nap M. Jornvall H. Schutte B. J. Pathol. 1999; 187: 567-572Crossref PubMed Scopus (567) Google Scholar). Positively stained cells were visualized with mouse-on-mouse immunodetection (Vector Laboratories, Burlingame, CA) and 3,3′ diaminobenzidine (Fast DAB, Sigma).To visualize mast cells, frozen sections were air-dried for ∼30 min, post-fixed in ice-cold acetone for 10 min, rinsed in PBS, and then stained in either Toluidine blue (a metachromatic dye for mast cells, made up as 0.1% Toluidine blue O (Sigma) in 1% sodium chloride, pH 2.3), Csaba stain (Alcian blue/Safranin O, an amine/heparin stain for mast cells, made up as 0.36% Alcian blue (Sigma), 0.018% Safranin O (Sigma) in acetate buffer, pH 1.42), or by using an enzymatic reaction with naphthol AS-D chloroacetate esterase (Sigma) to detect chymase activity (16Moloney W.C. McPherson K. Fliegelman L. J. Histochem. Cytochem. 1960; 8: 200-207Crossref PubMed Scopus (288) Google Scholar), then counterstained with Gill's hematoxylin or DAPI. For inhibitor-localization, frozen sections were prepared as above, then incubated overnight with 50 nm ecotin-PKal (10Stoop A.A. Craik C.S. Nat. Biotechnol. 2003; 21: 1063-1068Crossref PubMed Scopus (55) Google Scholar) biotinylated with Sulfo-NHS-LC-LC biotin (EZ-Link, Pierce), followed by 1-h incubation with Alexa 488-conjugated streptavidin (Invitrogen). Plasma kallikrein immunohistochemistry was performed on paraffin sections following sodium citrate antigen retrieval using a polyclonal antibody against recombinant mouse KLKB1 (R&D Systems) 1:50 overnight at 4 °C followed by a 30 min incubation with biotinylated anti-goat IgG secondary antibody (Amersham Biosciences) visualized using Vectastain ABC (Vector Laboratories) and 3,3′ diaminobenzidine (Fast DAB, Sigma).Histological and immunochemical images were acquired at 100× or 200× using a Leica DMR microscope and Leica Fire-Cam with accompanying software. Images were then imported into Adobe Photoshop software for analysis. Quantification of apoptotic cells was performed by counting the number of CytoDEATH+ cells in lumen spaces per 100× field. Quantification of lipid content was performed by measuring the number of Oil red O+ pixels per 100× field and expressing this as a percentage of the total field. Quantification of collagen was performed by measuring the number of collagen+ pixels per 200× field and expressing this as a percentage of the total field. A minimum of 4 independent images per animal in each group was analyzed for each assay.Real-time Quantitative Polymerase Chain Reaction—Total RNA was isolated from thoracic mammary glands cleared for muscle and lymph tissue at 3 weeks, 5 weeks, 15 days pregnant, 10 days lactating, and 4 days involuted CD1 mice. Liver tissue was used as a positive control. RNA isolation was performed using the RNA-Bee (Tel-Test, Inc., Friendswood, TX) phenol/guanidine thiocyanate/chloroform method for RNA isolation and concentration of RNA was measured spectrophotometrically. 5 μg of total RNA was used to perform reverse transcriptase polymerase chain reaction using Superscript II reverse transcriptase oligo(dT) reagents (Invitrogen). cDNA products were electrophoresed in 1% agarose/TAE gels to check thoroughness of the reverse transcriptase reaction.Real-time quantitative polymerase chain reaction (RT-qPCR) was performed by the Biomolecular Resource Center at the University of California, San Francisco. Klkb1 gene expression was normalized against expression of hypoxanthine phosphoribosyltransferase (HPRT), and reported as a RQ score relative to Klkb1 expression in 5-week-old mammary glands. Oligonucleotide primers and TaqMan probe used were as follows: Klkb1 (1272): forward primer: 5′-TGGTCGCCAATGGGTACTG-3′; Klkb1 (1342): reverse primer: 5′-ATATACGCCACACATCTGGATAGG-3′; Klkb1 probe: 5′-(FAM)-CAGCTGCCCATTGCTTTGATGGAATT-(BHQ1)-3′.PCR was conducted in triplicate with 20-μl reaction volumes of TaqMan Universal PCR Master Mix (Applied Biosystems), 0.9 mm of each primer, 250 nm probe, and 5 μl of cDNA. The PCR reaction was performed under the following conditions: 95 °C, 10 min, 1 cycle; 95 °C, 15 s then 60 °C, 2 min, 40 cycles. Analysis was carried out using the sequence detection software (SDS 2.1) supplied with TaqMan 7900HT (Applied Biosystems).RESULTSEcotin-PKal Is Specific for Human and Mouse Plasma Kallikrein—Previous work conducted in this laboratory suggested that plasma kallikrein is a regulator of in vivo adipogenesis in the mammary gland during involution (4Selvarajan S. Lund L.R. Takeuchi T. Craik C.S. Werb Z. Nat. Cell Biol. 2001; 3: 267-275Crossref PubMed Scopus (128) Google Scholar). However, the ecotin and ecotin variant (ecotin RR) used in the previous study target numerous serine proteases, including uPA in addition to PKal (17Yang S.Q. Craik C.S. J. Mol. Biol. 1998; 279: 1001-1011Crossref PubMed Scopus (22) Google Scholar). Therefore, we used a recently developed variant of ecotin that has picomolar specificity for PKal (ecotin-PKal), while having Ki* values four to seven orders of magnitude higher for related serine proteases. Importantly, the Ki*ofecotin-PKal for human plasmin is six orders of magnitude higher than the Ki* for human PKal, and seven orders of magnitude higher for the human plasminogen activators uPA and tPA (10Stoop A.A. Craik C.S. Nat. Biotechnol. 2003; 21: 1063-1068Crossref PubMed Scopus (55) Google Scholar). The only other protease that ecotin-PKal inhibits with reasonable (260-fold less) effectiveness is human Factor XIIa (10Stoop A.A. Craik C.S. Nat. Biotechnol. 2003; 21: 1063-1068Crossref PubMed Scopus (55) Google Scholar). This inhibition does not interfere with our investigation of the effects of PKal activity as PKal interacts with Factor XIIa as part of a reciprocal activation loop. Ecotin-PKal binding to serine proteases follows slow-tight binding kinetics (18Eggers C.T. Wang S.X. Fletterick R.J. Craik C.S. J. Mol. Biol. 2001; 308: 975-991Crossref PubMed Scopus (34) Google Scholar).To validate specificity in our mouse model system, binding titrations against mouse plasmin and mouse PKal were performed using ecotin-PKal as a ligand and measuring remaining (uninhibited) enzyme activity using p-nitroanilide substrates (Fig. 1A). The apparent affinities (Ki) of ecotin-PKal for mouse PKal and mouse plasmin were determined to be 40 nm and 60 μm, respectively. The affinity of ecotin-PKal for mouse PKal is still 1000-fold greater than its affinity for mouse plasmin, although less than for human PKal. Therefore, to inhibit PKal in vivo during mammary gland involution, we estimated that in our dosing regimen, peak circulating concentrations of ecotin-PKal in mouse plasma approach 2 μm, nearly 30-fold less than the apparent Ki for ecotin-PKal inhibition of plasmin. 3C. Craik, unpublished data. Thus, the effects of ecotin-PKal administration are due to the inhibition of mouse PKal and not of mouse plasmin in our model system.We collected mammary glands from 4-day involuting mammary glands, when PKal is highly expressed in these tissues (see below), to determine whether ecotin-PKal can bind active PKal from mammary tissue lysate. Using biotinylated ecotin-PKal-bound streptavidin beads, we pulled down PKal from mammary gland lysates (Fig. 1B). The bands indicated on the blot represent the full-length protein (∼80 kDa), and in its active form under reducing conditions, PKal heavy (∼42 kDa) and light (∼28 kDa) chains, which have at least one and two identified N-glycosylated Asn residues, respectively, according to the SwissProt Protein Data base. Similar results were obtained from 5-week-old virgin mammary gland lysates (data not shown). These data confirm that ecotin-PKal binds PKal found in the mammary gland.Mouse Plasma Kallikrein Is Differentially Expressed in the Developing Mammary Gland—Prekallikrein (gene: Klkb1), the precursor of PKal, is expressed in a variety of mouse and human tissues outside of the liver (19Cerf M.E. Raidoo D.M. Metab. Brain. Dis. 2000; 15: 315-323Crossref PubMed Scopus (16) Google Scholar, 20Ciechanowicz A. Bader M. Wagner J. Ganten D. Biochem. Biophys. Res. Commun. 1993; 197: 1370-1376Crossref PubMed Scopus (15) Google Scholar, 21Neth P. Arnhold M. Nitschko H. Fink E. Thromb. Haemost. 2001; 85: 1043-1047Crossref PubMed Scopus (29) Google Scholar). Accordingly, we wished to determine whether PKal is expressed in the mammary gland, and if its expression is altered during different phases of mammary gland development, when careful regulation of plasminogen activation is required.The process of pubertal mammary gland development begins around 3 weeks of age and continues until the advancing ductal epithelium reaches the end of the fat pad, around 8-10 weeks of age. We examined Klkb1 gene expression in the mouse mammary gland at different postnatal development time points (3 weeks, 5 weeks, 15 days pregnant, 10 days lactating, and 4 days involuting) by RT-qPCR. Klkb1 mRNA was abundant during pubertal development, when the mammary gland is undergoing active remodeling as the ductal epithelium expands and advances through the stromal fat pad (Fig. 2A). During pregnancy and lactation, when the stroma has largely been replaced by secretory alveoli and extensive ductal structures, Klkb1 mRNA was markedly reduced. Klkb1 mRNA increased significantly during mammary gland involution, when the secretory epithelial structures die by apoptosis and the mammary stromal compartment is replenished. These findings indicate that PKal is produced in the mammary gland, and that its increased expression levels correlate with periods of stromal remodeling.FIGURE 2Plasma kallikrein message is present in the mammary gland and the active protease localizes to mammary gland mast cells. A, real-time qPCR analysis of Klkb1 expression in mammary gland RNA shows that Klkb1 is differentially expressed throughout postnatal mammary gland development. RT-qPCR performed in triplicate for Klkb1 expression normalized against HPRT and reported as a RQ score relative to Klkb1 expression in 5-week-old mammary glands from CD1 mice. Increased expression correlates to periods of active stromal remodeling during mammary development. Error bars represent S.D. B, frozen section of 5-day-involuting mammary gland using Alexa 488-conjugated streptavidin (green) to visualize 50 nm bound biotinylated ecotin-PKal; DAPI counterstain (blue). White arrowheads indicate representative ecotin-PKal staining. C, staining is absent in presence of excess (50 μm) non-biotinylated ecotin-PKal. Scale bar for B-E is 100 μm. D, mast cells clustered around a large mammary blood vessel stain positively for active PKal when visualized using biotinylated ecotin-PKal. Frozen sections of 5-day-involuting mammary gland were stained with biotinylated ecotin-PKal as above. E, serial section of D stained for mast cells with Csaba stain shows that cells labeled by ecotin-PKal are mast cells. Black arrowheads indicate representative Csaba stained-mast cells. F, paraffin section of 5-week-old mammary gland shows mast cells in the mammary stroma stained with Toluidine blue, which stains mast cell granules purple. Sections were treated as a negative control for staining shown in G. Black arrowheads indicate Toluidine blue-stained mast cells. G, serial section of F immunostained with KLKB1 antibody followed by DAB shows that mammary gland mast cells stain positively for plasma kallikrein using conventional immunohistochemistry. Black arrowheads indicate Toluidine blue-stained, DAB+ mast cells. Scale bar for F and G is 100 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Biotinylated Ecotin-PKal Localizes Active Plasma Kallikrein to Mast Cells—Prekallikrein is expressed in the mammary gland in addition to circulating prekallikrein from the liver. However, it is active PKal that is inhibited by ecotin-PKal in vivo, resulting in repressed mammary gland involution. Therefore, we wished to identify which cells in the mammary gland are the sites of PKal activity. We used the biotinylated form of ecotin-PKal to localize the active protease in 5-day-involuting mammary gland tissue sections, taking advantage of ecotin-PKal's high affinity for the active form of PKal. Biotinylated ecotin-PKal stained a subset of cells in the mammary gland (Fig. 2B), and this staining was absent in the presence of a 1000-fold excess of non-biotinylated ecotin-PKa
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