Characterization of secretory leukocyte protease inhibitor as an inhibitor of implantation serine...

MOLECULAR REPRODUCTION AND DEVELOPMENT 75:1136–1142 (2008)

Characterization of Secretory LeukocyteProtease Inhibitor as an Inhibitor ofImplantation Serine ProteinasesNAVNEET SHARMA,1 JASPREET KAUR,2 HUI XU,1 NICOLE zur NIEDEN,1 AND DERRICK RANCOURT1*1Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary,Alberta, Canada2Department of Clinical Neuroscience, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada

ABSTRACT We have recently identified andcharacterized two implantation serine proteinasegenes, ISP1 and ISP2, which give rise to a dimericproteinase, ISP that facilitates embryo invasionduring peri-implantation period. As many proteinaseshave cognate serpins that regulate their proteolyticactivity, we have been investigating anti-tryptases,expressed during this window of implantation. Here,we report the differential expression of secretoryleukocyte protease inhibitor (SLPI) in uterine endome-trium around the implantation period. The co-local-ization of SLPI and ISP suggests the possibility thatSLPI is an ISP serpin and that expression of SLPImay lead to a reduction in ISP activity. The expressionof SLPI is down regulated during the window ofembryo-uterine receptivity. Our results are consistentwith a model suggesting that the drop in SLPIexpression may help to refine the opening of thewindow of implantation, by allowing the proteolyticactivity of embryo invasive serine proteinases suchas the ISPs. Mol. Reprod. Dev. 75: 1136–1142,2008. � 2007 Wiley-Liss, Inc.

Key Words: serpin; serine proteinase; implantation;fertility; endometrium

INTRODUCTION

Implantation, which is necessary for a successfulpregnancy, involves the coordination of embryonic anduterine factors (Carson et al., 2000). In the mouse modelof human implantation, a very short window of uterinereceptivity between days four and five post-coitum,requires a synchronization of events involving hor-mones and growth factors (Psychoyos, 1973). A varietyof proteinases have been postulated to be involved inthe process of embryo invasion including urokinaseplasminogen activator (uPA), matrix metalloproteinase9 (MMP9), and the cysteine proteinases, cathepsinB and cathepsin L. All of these proteinases have beenlocalized to the embryo-uterine boundary during theembryo invasion process (Alexander et al., 1996; Teesaluet al., 1996; Afonso et al., 1997).

There are a number of indicators suggestingthat these proteinases are tightly regulated. Firstly,

they may be acting in a cascade. uPA, for example, isresponsible for activating the ubiquitous proenzymeplasminogen into the active enzyme plasmin. Inturn, the uPA/plasmin system is thought to activateproMMP9 (Alexander et al., 1996). Secondly, proteolyticactivity may be localized to specific sub-cellular sites ofinvasion via receptors. uPA, for example, is bound byits receptor uPAR, at the site of invasion (Carrollet al., 1993; Teesalu et al., 1996). Finally, for each ofthese proteinases, there is a cognate anti-proteinase:plasminogen activator inhibitor (PAI), tissue inhibitormetalloproteinase 3 (TIMP3), and cytostatin C, that areeach localized to the uterine decidua surrounding theinvading embryo (Leco et al., 1996; Teesalu et al., 1996;Afonso et al., 2002).

We have identified two novel implantation serineproteinase genes, ISP1 and ISP2 (O’Sullivan et al.,2001a,b). Both genes are expressed in the preimplanta-tion embryo and the uterine endometrial glands duringimplantation (O’Sullivan et al., 2001a,b, 2002, 2004;Sharma et al., 2006). ISP protein and its proteolyticactivity are detectable in uterine fluid and are localizedto site of embryo invasion (O’Sullivan et al., 2004;Sharma et al., 2006). The cognate tryptase is minimallya hetero-dimer comprised of ISP1 and ISP2 peptides,although homo-dimers have not been ruled out (Sharmaet al., 2006). Synthetic inhibitors of ISP activity andanti-ISP antibodies have demonstrated a potential rolefor ISP in the early stages of embryo invasion in vitroand implantation in vivo (Huang et al., 2004; Sharmaet al., 2006).

Recently, secretory leukocyte protease inhibitor(SLPI), a broad spectrum serine proteinase inhibitorhas been reported in the uterine fluid of pigs and luminalepithelium of rats (Reed et al., 1998; Chen et al., 2004).As this serpin has been shown to have potent anti-tryptase activity (Wright et al., 1999), we sought to

� 2007 WILEY-LISS, INC.

Grant sponsor: Canadian Institute of Health Research (CIHR).

*Correspondence to: Derrick Rancourt, 3330 Hospital Dr NW,Calgary, AB, Canada T2N 4N1. E-mail: [email protected]

Received 30 July 2007; Accepted 10 October 2007Published online 28 December 2007 in Wiley InterScience(www.interscience.wiley.com).DOI 10.1002/mrd.20855

investigate the expression of SLPI in mouse andto investigate its potential role as an ISP anti-proteinase. Using immunohistochemistry, we showthat SLPI is expressed in both the luminal epitheliumand the endometrial glands throughout the peri-implantation period. When ISP is expressed during thewindow of implantation, it is co localized with SLPIin both the endometrial gland and within uterinefluid. Co-immunoprecipitation demonstrates that SLPIphysically interacts with both ISP1 and ISP2 mono-mers. Given that SLPI gene expression drops during thewindow of implantation, when ISP levels are at theirpeak, we suggest SLPI may help to refine the opening ofthe window by allowing the proteolytic activity ofembryo invasive serine proteinases such as the ISPs.

MATERIALS AND METHODS

Handling of Mice

CD1 mice were obtained at the age of 6–7 weeks fromCharles River Canada (St. Constant, Quebec) andmaintained in a standard laboratory animal facilitywith controlled temperature (208C) and lighting (lightson between 07:00 and 19:00 hr). The maintenance andtreatment of animals was in full compliance with theStandard laboratory animal care protocols approved byUniversity of Calgary’s Animal Care Committee. Toobtain natural pregnancies, female mice were pairedwith adult males and checked daily for the presence ofvaginal copulatory plug as an indication of mating. Forcollection of uteri/embryos, the pregnant mice weresacrificed on a specific day by cervical dislocation.

RT-PCR

Total RNA was prepared from the uteri of pregnantdams at different stages of pregnancy via Trizol lysis(Invitrogen, CA). The total RNA thus obtained wasreverse-transcribed using Superscript II (Invitrogen).The resulting cDNA was used as a template for PCR.Primer sequences:

SLPI (50-TGCTTAACCCTCCCAATGTC-30 and 50-G-AATGCTGAGCCAAAAGGAG- 30), ISP1 (50-GCGGAT-CCGTGGGGGAAGTA-30 and 50-GCGAATTCAGCTTT-GTGCTCG-30), ISP2 (50-GCGAATTCGTGGTACATCT-CC-30 and 50-GCGGATCCTATGGGGGCAA-30) weredesigned with the Primer3 program available at http://frodo.wi.mit.edu/cgibin/primer3/primer3_www.cgi andsubjected to a BLAST search (http://www.ncbi.nlm.nih.gov/blast). PCR reactions were done using 200 ng cDNAfrom each stage of pregnancy, in 25 ml reaction volume at558C melting temperature (Tm). The resulting DNAproducts were fractionated on 2% agarose gels at 70 V for45 min.

Real time PCR was carried out with 50 ng cDNA usinga two step PCR gradient in an iCycler iQ system using aSYBR green PCR master mix (Bio-Rad Labs, CA), withthe following cycle conditions: an initial denaturationstep at 958C followed by 40 rounds of cycling between30 sec at 958C and 45 sec at Tm (558C for SLPI and 568Cfor ISP1 and ISP2). Expression of SLPI and ISPs was

normalized to the house keeping gene, GAPDHand compared to expression levels obtained from non-pregnant uteri.

Immunofluorescence Microscopy

Paraffin sections (6 mm) of mouse uteri at differentstages of pregnancy were cleared in xylene and re-hydrated by passing them through different grades ofalcohol (100%, 95%, 75%, 50%, 30%) and finally tomilliQ H2O and PBS. Sections were subsequentlyblocked with 10% normal goat serum PBS for 1 hrat room temperature. Thereafter, the sections wereco-labeled for SLPI and ISP1/2 by incubating themwith anti-mouse SLPI rabbit polyclonal (Abcam; 1:200)and anti-ISP1 or anti-ISP2 monoclonal antibodies(1:200) overnight at 48C. Sections were then washedwith PBS three times for 5 min each to clear unboundantibody. Subsequent incubation of sections with goatanti-mouse-Cy3 (Zymed; 1:200) and goat anti-rabbit-FITC (Zymed; 1:200) fluorescent secondary antibodieswas done for 2 hr at room temperature. Sections werewashed thereafter in PBS (3� 5 min) and cover-slippedusing a mounting medium (Zymed, CA). Fluorescencewas detected using Olympus BX51 microscope. Image-Pro Plus software was used to acquire photomicro-graphs of the labeled sections. Green fluorescence(FITC) indicated the location of SLPI within mouseuterine sections, while red fluorescence (Cy3) indicatedthe localization of ISP1 or ISP2.

Immunoprecipitation and Immunoblotting

Immunoprecipitations (IPs) were carried out by usingProtein G beads and anti-mouse SLPI rabbit polyclonalantibody (above). Uterine tissue homogenates wereused in equivalent protein concentration (1.0 mg totalprotein) for each stage of pregnancy. After IP, the pulled-down proteins were detected by immunoblotting usingthe same antibody or anti-ISP1 or anti-ISP2 monoclonalantibodies. Immunoprecipitates were first separated by10% PAGE run under denaturing conditions. Proteinswere then transferred to nitrocellulose membraneand probed with the respective antibodies. An HRP-labeled anti-rabbit or anti-mouse IgG preparations (GEHealthcare, NJ) were used at a 1:10,000 dilution fordetection on BioMaxTM (Kodak, CT) film.

RESULTS

Dynamic and Opposing Expression of SLPIand ISP in Pregnant Mouse Uterus

As SLPI is a potent inhibitor of tryptases, we sought toinvestigate its potential role as an ISP serpin. In initialexperiments, we used RT-PCR to identify the presenceof SLPI transcripts in mouse uterus from Days 1.5 to8.5 around the window of implantation (Fig. 1A). Weobserved that SLPI expression is strong before and afterthe window of implantation, but is significantly depletedwhen the window is open on Days 4.5 and 5.5. Theseresults were confirmed by real time RT-PCR (Fig. 1B).Here, we observed that SLPI expression is highest

Molecular Reproduction and Development

SLPI IS AN ISP SERPIN 1137

on Day 0.5 but steadily declined to its lowest pointon Day 4.5, whereupon it began to slowly recover upuntil Day 8.5. This expression is sharply in contrast tothe expression of the ISP1 and 2 genes (Fig. 1C,D), whichwere abundantly expressed in mouse uterus during thewindow of implantation on Days 4.5 and 5.5, as we haveseen previously (O’Sullivan et al., 2001b, 2002).

In order to monitor SLPI expression at the proteinlevel, immunoblotting experiments were performedusing a commercial antibody and uterine tissue homo-genates obtained at different stages of pregnancy.However, very weak signals were obtained (data notshown), owing to a problem of SLPI abundance. As theantibody was successfully used in SLPI immunofluor-escence microscopy (Figs. 2 and 4), we attributed thisdifficulty to a lack of SLPI abundance in uterine tissueextracts. Indeed, when we carried out immunoprecipi-tations using large volumes of uterine tissue homoge-nates and hence larger amounts of protein content atdifferent stages of pregnancy, we were able to detectSLPI as a 12 kDa band on a western blot (Fig. 3A).However as immunoprecipitation is not a quantitativetechnique, it could not be used to confirm whetheruterine SLPI expression was dynamic at the proteinlevel.

Similarly differential expression of ISP1 and ISP2was validated at the protein level by carrying outimmunoblotting experiments using custom made mono-clonal anti-ISP1 and anti-ISP2 antibodies (Fig. 3C,D).The expression of ISPs is more pronounced at Days 4.5–6.5 validating the data obtained at transcriptional level(Fig. 1C,D).

SLPI and ISP Co-Localize in theEndometrial Glands

We used co-immunofluorescence to examine thepotential co-localization of SLPI (green signal) andISP1 or ISP2 (red signal) from Days 1.5 to 6.5 (Fig. 4).SLPI staining was mostly limited to luminal andglandular epithelium throughout this period. In con-trast, ISP staining was restricted to the endometrialglands, as described previously (O’Sullivan et al.,2001b, 2002). Although ISP staining became prevalenton Days 4.5 and 5.5 (Fig. 2D,E), evidence of ISP proteinexpression was observed as early as Days 2.5 and 3.5(Fig. 2B and C). At higher magnification, SLPI and ISPoften appeared to be co-localized within the lumen of theendometrial gland (Fig. 4A,C). Although SLPI stainingoccurred throughout the cytoplasm of the glandularcells, co-localization appeared to be restricted to basal


Fig. 1. Expression of SLPI and ISP (ISP1 and ISP2) transcripts at different stages of pregnancy,(A) RT-PCR for SLPI expression, (B) real time PCR data for SLPI expression at different stages ofpregnancy, (C) real time PCR data for ISP1 expression at different stages of pregnancy, (D) real time PCRdata for ISP2 expression at different stages of pregnancy. [See color version online at www.interscience.wiley.com.]

1138 N. SHARMA ET AL.

part of each glandular cell. On rare occasion we werealso able to detect co-localization within passages ofuterine lumen (Fig. 4B,D). In these circumstances aswell, co-localization is most prevalent at the base of theepithelium.

SLPI and ISP Physically Interact

With our ability to immunoprecipitate SLPI through-out the time period surrounding the window of implan-tation, we sought to investigate the possibility thatSLPI and ISP physically interacted. SLPI immuno-precipitates were subjected to 10% PAGE, and immuno-blotted using ISP1 and ISP2 monoclonal antibodies. Thepresence of the 63 kDa ISP dimer was revealed (Fig. 3B).These results in combination with co localization data(Fig. 4) point toward the close relationship betweenSLPI and ISP enzyme complex.

DISCUSSION

SLPI, also known as anti-leukoprotease, is a memberof the chelonianin class of the serine protease inhibitors

(Serpins) (Francart et al., 1997). It has a broad spectruminhibitory activity against mast cell and leukocyteserine proteases, including cathepsin G, elastase, andtryptase (Thompson and Ohlsson, 1986; Wright et al.,1999). It does not inhibit kallikrein, thrombin, factor10a, uPA, or plasmin. SLPI is primarily associatedwith a variety of secretory/glandular epithelial cellsincluding lung and cervix (De Water et al., 1986; Heinzelet al., 1986). Recently, it has been implicated as an anti-bacterial, -viral, and -fungal agent (Hiemstra et al.,1996; Shine et al., 1997; Tomee et al., 1997). SLPIknockout mice (Ashcroft et al., 2000) display a disrup-tion in wound healing consistent with an increase inelastase activity. They also display a prolonged inflam-matory response that is associated with the proteolyticcascade that disrupts prostaglandin production. Inter-estingly, disruption of SLPI had no effect on fertility.

Several serpins have been found as a part of the milieuof uterine fluid including plasminogen activator inhbi-tor-1, alpha-1 chymotrypsin, uterine plasmin/trypsininhibitor and SLPI (Badinga et al., 1994; Miyauchi et al.,


Fig. 2. Immunoflouresecnce microscopy studies showing expression of SLPI (green) and ISP1/ISP2 (red)in mouse uterus at different stages of pregnancy, (A) 1.5 dpc, (B) 2.5 dpc, (C) 3.5 dpc, (D) 4.5 dpc, (E) 5.5 dpc,(F) 6.5 dpc.


1995; King et al., 2000). SLPI has been found in theendometrium and myometrium of pregnant pigs, cowsand horses, where it has been implicated in epithelio-chorial placentation (Helmig et al., 1995). However,SLPI is also associated with hemochorial placentation(i.e., implantation). In humans, SLPI expression occursin the first trimester of pregnancy and has been detectedin the crypts of endocervical glands (Heinzel et al.,1986). In rat, SLPI RNA expression has only beendetected in the luminal epithelium using in situhybrid zation (Chen et al., 2004). Using immunohisto-chemistry, we have observed SLPI protein in boththe endometrial gland and in luminal epithelium.Indeed our expression results suggest other differencesbetween rat and mouse. Whereas others have reportedthat rat SLPI RNA level are induced by estrogen anddissipate after the window of implantation (Chen et al.,2004), we have observed that SLPI RNA expression inpregnant mouse uterus diminishes before the windowopens on Day 4 (Fig. 1). It is when the window opens

on Days 4 and 5, when we observe maximal expression ofthe tryptase genes ISP1 and ISP2 in pregnant uterus.

We have previously demonstrated that the ISP1 andISP2 gene products dimerize to give rise to a tryptasepresent in both the preimplantation embryo and uterinefluid and involved in the processes of embryo hatchingand the initiation of implantation (Sharma et al., 2006).Knowing SLPI’s anti-tryptic activity and its diminish-ing expression during the rise of ISP expression, wehypothesize that SLPI may be acting as an ISP anti-proteinase. Although we have been unable to obtainmouse SLPI in order to test its anti-ISP potential, wehave observed that ISP and SLPI purify as a complex(Fig. 3) and co-localize within endometrial gland lumenand basal lamina and to specific regions within theluminal epithelium (Fig. 4).

This interaction between SLPI and ISP occursthroughout the peri-implantation period and we specu-late that it may play a role in regulating ISP proteolyticactivity. For example, SLPI expression in the glandular


Fig. 3. Expression of SLPI and ISPs (ISP1 and ISP2) at differentstages of pregnancy, (A) immunoblot showing IP of SLPI by anti-SLPIantibodies from uterine tissue homogenates at different stages ofpregnancy, lane 1—nonpregnant, lane 2—0.5 dpc, lane 3—1.5 dpc,lane 4—2.5 dpc, lane 5—3.5 dpc, lane 6—4.5 dpc, lane 7—5.5 dpc,lane 8—6.5 dpc, lane 9—7.5 dpc, lane 10—8.5 dpc; (B) immunoblotshowing co-IP of ISP1-ISP2 enzyme complex (63 kDa) at differentstages of pregnancy by anti-SLPI antibodies, lane 1—nonpregnant,

lane 2—0.5 dpc, lane 3—1.5 dpc, lane 4—2.5 dpc, lane 5—3.5 dpc,lane 6—4.5 dpc, lane 7—5.5 dpc, lane 8—6.5 dpc, lane 9—7.5 dpc, lane10—8.5 dpc; (C,D) immunoblots showing differential expression ofISP1 (C) and ISP2 (D) found in the uterine tissue homogenates atdifferent stages of pregnancy, lane 1—nonpregnant, lane 2—0.5 dpc,lane 3—1.5 dpc, lane 4—2.5 dpc, lane 5—3.5 dpc, lane 6—4.5 dpc,lane 7—5.5 dpc, lane 8—6.5 dpc, lane 9—7.5 dpc, lane 10—8.5 dpc,lane 11—9.5 dpc, lane 12—10.5 dpc.


epithelium may play a role in preventing proteolyticactivity before the implantation window opens. Wehave previously suggested that hormonal regulation isimportant for preventing ISP translation prior to theopening of the window of implantation. Althoughrising progesterone levels on Day 2 are sufficient toinduce ISP1 and ISP 2 mRNA transcription, increasingestrogen levels on Day 3 have the opposite effectand prevent the translation of ISP1 and ISP2 mRNA(O’Sullivan et al., 2004). This estrogen spike, whichsynchronizes embryo and uterine receptivity and opensthe window, has the net effect of allowing the accumu-lation of ISP1 and ISP2 transcripts, so that a burst of ISPis produced after the estrogen dissipates and when theembryo has entered the uterus.

Although we have previously observed small amountsof ISP in uterine fluid before the estrogen spike we havereconciled that the presence of a small amount ofenzyme is inconsequential when the embryo is still inthe oviduct. However, as we have observed in thisreport, a small amount of glandular ISP expression isalso detectable on Day 3 (Fig. 1C) when the embryohas reached the uterus. Hence, the presence of SLPI atthis time may help to inhibit the limited amount ofactive enzyme. In turn, it is only when ISP levelsrise significantly above circulating levels of SLPIduring Days 4.5–5.5, that the ISP mediated initiationof implantation is allowed to occur. This may be a rathersimplistic view of the role of SLPI in regulating ISP andmore sophisticated mechanisms can be envisioned.More experiments will be required to confirm whetherSLPI indeed has anti-ISP activity at the biochemical

level or perhaps plays some other role through thisinteraction.

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