BIOLOGY OF REPRODUCTION 61, 380±387 (1999) Uterine-Associated Serine Protease Inhibitors Stimulate Deoxyribonucleic Acid Synthesis in Porcine Endometrial Glandular Epithelial Cells of Pregnancy 1 Lokenga Badinga, Frank J. Michel, and Rosalia C.M. Simmen2 Animal Molecular and Cell Biology Interdisciplinary Concentration, Department of Animal Science, University of Florida, Gainesville, Florida 32611-0910 ABSTRACT Consistent with this, uteri from mammalian species with distinct placentation types express common classes of pro- Protease inhibitors are major secretory components of the tease inhibitors (e.g., tissue inhibitors of metalloproteases, Downloaded from https://academic.oup.com/biolreprod/article/61/2/380/2734487 by guest on 24 September 2021 mammalian uterus that are thought to mediate pregnancy-as- TIMPs) as well as distinct ones (e.g., secretory leukocyte sociated events primarily by regulating the activity of proteolytic protease inhibitor, SLPI, and uterine plasmin/trypsin inhib- enzymes. In the present study, we examined the mitogenic po- tentials of two serine protease inhibitors, namely secretory leu- itor, UPTI) [4, 8, 9]. Since embryos from all species, re- kocyte protease inhibitor (SLPI) and uterine plasmin/trypsin in- gardless of placentation type, exhibit invasive properties hibitor (UPTI) in primary cultures of glandular epithelial (GE) when placed into ectopic sites [10], the limiting of blasto- cells isolated from early pregnant (Day 12) pig endometrium, cyst invasiveness, albeit to varying extents, is most likely using the [3H]thymidine incorporation assay. Puri®ed porcine an important function of pregnancy-associated uterine anti- SLPI (pSPLI), porcine UPTI (pUPTI), or recombinant human SLPI proteases. (rhSLPI), all of which exhibited anti-trypsin activity, increased The recent demonstration that TIMPs 1 and 2 exhibit (p , 0.05) labeled thymidine incorporation into DNA of serum- growth-promoting activity in various mammalian cell types, deprived GE cells when tested at a range of 10±1000-ng/ml con- independent of their anti-proteolytic activity, points to a centrations. Polyclonal antibodies directed against either hSLPI potentially novel function(s) for anti-proteases in pregnancy or pSLPI abrogated the effect of SLPI. Co-addition of pSLPI and events [11±13]. The mechanism(s) underlying the growth pUPTI increased DNA synthesis in these cells to a level higher factor activity of TIMPs is presently unclear; however, re- (p , 0.05) than that observed with either protease inhibitor. The cent studies have indicated that this may occur through spe- glycosaminoglycan heparin, which has been previously shown ci®c cell surface receptors [13], similar to pathways utilized to increase the anti-protease activity of SLPI, exhibited a ten- by classical growth factors. The possibility that distinct cell dency (p 5 0.08) to enhance SLPI and UPTI induction of cellular types of the uterine endometrium represent target sites for DNA synthesis. Reverse transcription-polymerase chain reaction the growth-promoting action of TIMPs or any other pro- indicated that the messenger RNAs for both protease inhibitors tease inhibitors has not been previously explored. were present in the endometrium throughout pregnancy and, The pig uterus of pregnancy synthesizes several low- within this tissue, in GE cells to a greater extent (p , 0.05) than molecular-weight anti-proteases, which include SLPI [8], in stromal ®broblastic cells. Results demonstrate that, in addi- tion to their well-documented anti-protease activities, SLPI and UPTI [9], and TIMPs 1 and 2 [14]. The apparent restricted UPTI may constitute autocrine growth promotants for the uter- uterine expression of SLPI and UPTI, but not of TIMPs, to ine epithelium. These data suggest a novel mechanism whereby species with noninvasive placentation, implicates their locally produced protease inhibitors may modulate periimplan- function in the maintenance of an intact utero-placental in- tation events and embryo-maternal communication. terface [15, 16]. Additionally, unlike that of TIMPs, ex- pressions of SLPI and of UPTI in the pig uterine endo- INTRODUCTION metrium are induced by the presence of periimplantation conceptuses, and more speci®cally for SLPI, by their se- The mammalian uterus of early pregnancy is a major site cretory products [14, 17]. This modulation, a form of em- of synthesis for different classes of protease inhibitors [1± bryo-maternal communication, probably represents a mech- 4]. This high level of production probably represents a ma- anism that potentiates the uterine functions of SLPI and jor defense mechanism through which it fends off proteases UPTI to positively affect early pregnancy events. The na- that originate from within itself, the blastocyst, the feto- ture of these uterine function(s), distinct from inhibition of placental unit, or in®ltrating neutrophils and mast cells that protease activity, has not been elucidated. accompany implantation events [5, 6]. Although the action The present study examined whether uterine-derived of proteases is essential for uterine tissue remodeling and SLPI and UPTI, similar to TIMPs 1 and 2, exhibit addi- the initiation of interaction with the implanting conceptus, tional functions distinct from their protease inhibitor activ- this activity must be carefully controlled. Thus, a biological ities and, if so, whether these effects occur in an autocrine rationale exists for the presence of a wide variety of pro- manner. Towards this end, the mitogenic potentials of pu- tease inhibitors with overlapping substrate speci®cities ri®ed preparations of these porcine uterine proteins, alone within the uterine micro-environment. Indeed, it has been or in combination, was tested in vitro, using primary cul- suggested that the extent of embryo-maternal interaction, tures of glandular epithelial cells isolated from endometri- and hence the nature of placentation, is dependent upon the um of early pregnancy. levels and types of proteases and protease inhibitors [7]. MATERIALS AND METHODS Accepted March 9, 1999. Received November 12, 1998. Materials 1This work was supported in part by USDA grants 94-37205-1164 and 96-35205-3745, and NIH HD21961 to R.C.M.S. This is Journal Series No. The polymerase chain reaction (PCR) optimizer kit and R-06687 from the Florida Agricultural Experiment Station. cDNA cycle kit were purchased from Invitrogen (San Di- 2Correspondence. FAX: 352 392 7652; e-mail: [email protected]¯.edu ego, CA). Taq DNA polymerase was obtained from Boeh- 380 UTERINE PROTEASE INHIBITORS AS EPITHELIAL MITOGENS 381 ringer Mannheim (Indianapolis, IN). [a-32P]Deoxycytidine gel and visualized by ethidium bromide staining. In some triphosphate (SA 3000 Ci/nmol) and BioTrans nylon mem- experiments, the products were blotted onto nylon mem- branes (0.2 mm) were purchased from ICN Radiochemicals branes and analyzed by Southern blotting, using the pUPTI (Irvine, CA), and the nick-translation kit was from Amer- cDNA probe. For analysis of PCR band intensities, pho- sham Corp (Arlington Heights, IL). TRIzol and culture me- tographs of ethidium bromide-stained gels or autoradio- dia were from GIBCO BRL (Gaithersburg, MD). Trypsin grams were scanned at high resolution, and the integrated (type XIII, L-1-tosylamino-2-phenylethylchloramethyl ke- density of the band was calculated using the Alpha Imager tone-treated), trypsin inhibitor (type I-S), N-benzoyl-DL-ar- 2000 Documentation & Analysis System (Alpha Innotech ginine-p-nitroanilide, and heparin were purchased from Sig- Corp., San Leandro, CA). The intensities of the SLPI and ma Chemical Co. (St. Louis, MO). Recombinant human UPTI signals were normalized to that of the b2-microglob- SLPI (rhSLPI) was obtained from R&D Systems Inc (Min- ulin internal control. The PCR-generated fragments were neapolis, MN). also puri®ed from primers using PCR wizard prep columns (Promega, Madison, WI) and ligated into the TA cloning Cell Culture and RNA Extraction vector pCR2.1 (Invitrogen) to isolate pUPTI and pSLPI Downloaded from https://academic.oup.com/biolreprod/article/61/2/380/2734487 by guest on 24 September 2021 clones for nucleotide sequence analyses. The cDNA clones Uterine GE and stromal (ST) cells were isolated from were sequenced in both directions by the Sanger dideoxy- Day 12 pregnant pig endometrium as previously described 6 nucleotide chain termination method [20]. Sequence infor- [18]. Cells were resuspended (0.5 3 10 cells/ml) in RPMI mation was analyzed using the Sequence Analysis Software 1640 medium containing 10% heat-inactivated fetal bovine package from the Genetics Computer Group (Madison, serum and 0.25 U/ml insulin, and cultured at 378Cina95% WI). The entire nucleotide sequences of pSLPI (500 bp) air:5% CO2 environment. Cells remained undisturbed (i.e., and pUPTI (479 bp) PCR fragments (data not shown) were without medium change) for the ®rst three days after plat- 100% identical to those previously reported [8, 15]. ing. Thereafter, each culture was replenished with fresh RPMI 1640 containing serum every two days until cells reached 100% con¯uence. Total cellular RNA was isolated Northern Blot Analysis from con¯uent cells using TRIzol reagent according to the manufacturer's instructions. After chloroform extraction, Total cellular RNA from porcine endometrial tissue was RNA was recovered from the aqueous phase and precipi- extracted by the guanidinium thiocyanate-phenol-chloro- tated with isopropanol. The RNA pellet was dissolved in form method. Thirty micrograms of