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a Animal Molecular and Cell Biology Interdisciplinary Concentration, Department of Animal Science, University of Florida, Gainesville, Florida 32611-0910
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The recent demonstration that TIMPs 1 and 2 exhibit growth-promoting activity in various mammalian cell types, independent of their anti-proteolytic activity, points to a potentially novel function(s) for anti-proteases in pregnancy events [11–13]. The mechanism(s) underlying the growth factor activity of TIMPs is presently unclear; however, recent studies have indicated that this may occur through specific cell surface receptors [13], similar to pathways utilized by classical growth factors. The possibility that distinct cell types of the uterine endometrium represent target sites for the growth-promoting action of TIMPs or any other protease inhibitors has not been previously explored.
The pig uterus of pregnancy synthesizes several low-molecular-weight anti-proteases, which include SLPI [8], UPTI [9], and TIMPs 1 and 2 [14]. The apparent restricted uterine expression of SLPI and UPTI, but not of TIMPs, to species with noninvasive placentation, implicates their function in the maintenance of an intact utero-placental interface [15, 16]. Additionally, unlike that of TIMPs, expressions of SLPI and of UPTI in the pig uterine endometrium are induced by the presence of periimplantation conceptuses, and more specifically for SLPI, by their secretory products [14, 17]. This modulation, a form of embryo-maternal communication, probably represents a mechanism that potentiates the uterine functions of SLPI and UPTI to positively affect early pregnancy events. The nature of these uterine function(s), distinct from inhibition of protease activity, has not been elucidated.
The present study examined whether uterine-derived SLPI and UPTI, similar to TIMPs 1 and 2, exhibit additional functions distinct from their protease inhibitor activities and, if so, whether these effects occur in an autocrine manner. Towards this end, the mitogenic potentials of purified preparations of these porcine uterine proteins, alone or in combination, was tested in vitro, using primary cultures of glandular epithelial cells isolated from endometrium of early pregnancy.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The polymerase chain reaction (PCR) optimizer kit and cDNA cycle kit were purchased from Invitrogen (San Diego, CA). Taq DNA polymerase was obtained from Boehringer Mannheim (Indianapolis, IN). [
-32P]Deoxycytidine triphosphate (SA 3000 Ci/nmol) and BioTrans nylon membranes (0.2 µm) were purchased from ICN Radiochemicals (Irvine, CA), and the nick-translation kit was from Amersham Corp (Arlington Heights, IL). TRIzol and culture media were from GIBCO BRL (Gaithersburg, MD). Trypsin (type XIII, L-1-tosylamino-2-phenylethylchloramethyl ketone-treated), trypsin inhibitor (type I-S), N-benzoyl-DL-arginine-p-nitroanilide, and heparin were purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant human SLPI (rhSLPI) was obtained from R&D Systems Inc (Minneapolis, MN).
Cell Culture and RNA Extraction
Uterine GE and stromal (ST) cells were isolated from Day 12 pregnant pig endometrium as previously described [18]. Cells were resuspended (0.5 x 106 cells/ml) in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum and 0.25 U/ml insulin, and cultured at 37°C in a 95% air:5% CO2 environment. Cells remained undisturbed (i.e., without medium change) for the first three days after plating. Thereafter, each culture was replenished with fresh RPMI 1640 containing serum every two days until cells reached 100% confluence. Total cellular RNA was isolated from confluent cells using TRIzol reagent according to the manufacturer's instructions. After chloroform extraction, RNA was recovered from the aqueous phase and precipitated with isopropanol. The RNA pellet was dissolved in sterile water and quantified by spectrophotometry.
Reverse Transcription (RT)-Polymerase Chain Reaction (PCR), Southern Blot, and Sequence Analyses of PCR Products
Five micrograms each of total RNA isolated from GE and ST cells were reverse-transcribed as described previously [19]. Each RT reaction mixture was extracted with phenol-chloroform and then precipitated with ethanol at -80°C overnight. The cDNA pellet was recovered by centrifugation, resuspended in 20 µl of sterile water, and frozen at -20°C until analyzed.
The PCR analysis was subjected to preliminary validation and optimization steps to ensure that the assay was linear relative to the amount of input RNA and that amplification of the products was in the exponential phase. These steps included 1) determining the optimal Mg2+ concentration and pH for each DNA template-primer combination, using the PCR optimizer kit; 2) varying the template concentration, once the optimal buffer was identified; and 3) adjusting the number of cycles under conditions of optimal buffer and template amount. For both SLPI and UPTI, the determined optimal PCR conditions were 1) 2 min at 95°C, 1.5 min at 55°C, and 2 min at 72°C; 35 cycles; 2) buffer G (60 mM Tris-HCl [pH 9.0], 15 mM ammonium sulfate, 2.5 mM MgCl2); and 3) 0.3 µg of each primer and 2 µl of diluted (1:10) RT reaction. The sequences of gene-specific oligonucleotide primers were designed from the published cDNA sequences of porcine SLPI (pSLPI) and porcine UPTI (pUPTI), respectively [8, 15], and were as follows: 1) pSLPI, forward: 5'-CGTGGAAGGTGCTGAAAATG-3'; reverse: 5'-CATGTCCTGGAAGCCTACAG-3'; and 2) pUPTI, forward: 5'-AGCCCTCCTCCTCCTCCTGG-3'; reverse: 5'-CACTCAGAATGGGATGTCGA-3'. The expected product sizes were 500 base pairs (bp) and 479 bp, respectively, for pSPLI and pUPTI. Aliquots of the PCR reactions (15 µl) were electrophoresed in a 1.5% agarose gel and visualized by ethidium bromide staining. In some experiments, the products were blotted onto nylon membranes and analyzed by Southern blotting, using the pUPTI cDNA probe. For analysis of PCR band intensities, photographs of ethidium bromide-stained gels or autoradiograms were scanned at high resolution, and the integrated density of the band was calculated using the Alpha Imager 2000 Documentation & Analysis System (Alpha Innotech Corp., San Leandro, CA). The intensities of the SLPI and UPTI signals were normalized to that of the ß2-microglobulin internal control. The PCR-generated fragments were also purified from primers using PCR wizard prep columns (Promega, Madison, WI) and ligated into the TA cloning vector pCR2.1 (Invitrogen) to isolate pUPTI and pSLPI clones for nucleotide sequence analyses. The cDNA clones were sequenced in both directions by the Sanger dideoxynucleotide chain termination method [20]. Sequence information was analyzed using the Sequence Analysis Software package from the Genetics Computer Group (Madison, WI). The entire nucleotide sequences of pSLPI (500 bp) and pUPTI (479 bp) PCR fragments (data not shown) were 100% identical to those previously reported [8, 15].
Northern Blot Analysis
Total cellular RNA from porcine endometrial tissue was extracted by the guanidinium thiocyanate-phenol-chloroform method. Thirty micrograms of total RNA was subjected to Northern blot analysis as previously described [8]. Membranes were hybridized with 32P-labeled pSLPI or pUPTI cDNA fragments, according to previously described procedures [8].
Purification of Porcine SLPI and Porcine UPTI
Samples of allantoic fluid (AF) collected from Day 60 pregnant pigs were clarified by low-speed centrifugation, and proteins were precipitated with 80% ammonium sulfate at 4°C for 1 h. The pellet was recovered by centrifugation (50 000 x g) for 20 min and redissolved in 20 mM Tris-HCl buffer (pH 8.0) to a final volume of 32 ml/500 ml of starting volume of AF. Basic proteins were enriched by allowing them to bind to carboxymethyl (CM) cellulose (Bio-Rad, Richmond, CA) and were then eluted with 20 mM Tris-HCl buffer (pH 8.0) over a 0–1 M linear NaCl gradient. Fractions were evaluated for the presence of SLPI by a specific RIA, following protocols previously described [21] and by a trypsin inhibition assay (see below). The active fractions were pooled and then size-fractionated by gel filtration over a Sephadex G-50 column previously equilibrated with Tris buffer (20 mM, pH 8.0), at a flow rate of 40 ml/h. The eluted fractions were assayed for SLPI immunoreactivity and anti-trypsin activity, respectively; those exhibiting peak activities in both assays were examined for purity by SDS-PAGE and then stained with Coomassie blue or silver dye. Highly purified fractions were pooled and subjected to NH2-terminal amino acid sequencing (see below).
During the purification of pSLPI, fractions exhibiting anti-trypsin activity but lacking immunoreactivity towards anti-SLPI antiserum, were identified from CM cellulose chromatography. These fractions were pooled and subjected to further purification by size-fractionation over a Sephadex G-50 column previously equilibrated with Tris buffer (20 mM, pH 8.0). The fractions exhibiting highest anti-trypsin activity were evaluated for purity by SDS-PAGE. The final confirmation of the purified protein's identity as UPTI was determined by NH2-terminal amino acid analysis.
Amino Acid Sequencing
Active fractions containing SLPI and UPTI, respectively, were separated on a one-dimensional SDS-PAGE gel, and proteins were electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes by the method of Towbin [22]. The membranes were rinsed in water, stained with 0.01% Coomassie Blue dye in 50% methanol, and destained in 50% methanol. The protein bands of desired molecular weights were sequenced using an Applied Biosystems (Foster City, CA) model 470A Gas Phase Protein Sequencer with an on-line analytical HPLC system, at the Interdisciplinary Center for Biotechnology Research Protein Core, University of Florida.
Trypsin Inhibition Assay
The ability of pSLPI, pUPTI, and rhSLPI to inhibit the enzymatic activity of trypsin was examined according to previously described protocols [15]. Briefly, known concentrations of these proteins, as measured by the Bio-Rad assay (Bio-Rad Technical Bulletin) were incubated with 100 µl of trypsin (20 µg/ml) at room temperature for 15 min. The total volume of the reaction was adjusted to 500 µl by addition of 250 µl of 50 mM Hepes buffer (pH 7.0) and 50 µl of 50 mM N-benzoyl-DL-arginine-p-nitroanilide. After an additional 25-min incubation at 37°C, the reaction was terminated by the addition of 500 µl of soybean trypsin inhibitor (10 µg/ml), and the absorbance at 410 nm was measured. The inhibitory activity in each test sample was expressed as a percentage of the residual trypsin activity, with the activity of trypsin in the absence of added SLPI or UPTI considered to be 100%.
[3H]Thymidine Incorporation into Cellular DNA
Mitogenic responses of uterine endometrial GE cells to SLPI and UPTI, alone or in combination, and in the presence or absence of heparin were examined via the incorporation of [3H]thymidine into DNA as previously described [23]. Confluent GE cells were washed twice in Hanks' Balanced Salt Solution and preconditioned in serum-free medium for 24 h. Medium was then changed, and the cells were incubated with or without the indicated protease inhibitors for an additional 24 h. At 4 h before the end of the incubation period, cells were pulse-labeled with 2 µCi of [3H]thymidine. Cells were rinsed with PBS (pH 7.4), DNA was precipitated with 5% trichloroacetic acid, and incorporated radioactivity was measured by liquid scintillation counting.
Statistical Analysis
Differences in mitogenic responses due to protease inhibitor and/or heparin treatments and numerical data from densitometric analysis of PCR or hybridization bands were examined by least-squares ANOVA using the General Linear Models procedure of the Statistical Analysis System [24]. The effects of protease inhibitors were analyzed using a mathematical model that included treatment (control, SLPI, UPTI, SLPI + UPTI), pig, and treatment x pig interaction. The dose effects were tested using a statistical model that included dose, pig, and pig x dose interaction. In both mathematical models, the pig effect was analyzed as a random effect, and the interactions between treatment x pig or dose x pig were used to test the fixed effects of treatment and dose, respectively. The mathematical model for heparin effect included treatment (control vs. heparin), protease inhibitor (control, SLPI, UPTI, SLPI + UPTI), treatment x protease interaction, pig, treatment x pig interaction, protease inhibitor x pig interaction, and treatment x protease inhibitor x pig interaction. Effects of pig and interactions involving pig were analyzed as random effects and used as error terms for appropriate upstream fixed effects. Significant effects (p < 0.05) of dose or treatments were separated by preplanned orthogonal contrasts. Results represent means ± SEM of two to three independent experiments, with each experiment representing 3 separate cultures of endometrial GE cells isolated from an individual gilt at Day 12 of pregnancy.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Porcine SLPI and UPTI were purified from Day 60 pregnant pig AF by successive gel filtration over CM cellulose and Sephadex G-50 columns, respectively. In each case, eluted fractions containing the desired proteins were identified by evaluating their reactivity towards anti-SLPI antiserum by RIA [21] and by their ability to inhibit trypsin. Fractions corresponding to peak activity were further examined for homogeneity by one-dimensional SDS-PAGE. The purified pSLPI protein used in all in vitro assays contained two bands migrating slightly below the Mr 20 000 and above the Mr 14 200 standards (Fig. 1A). NH2-terminal amino acid sequence analyses of these two proteins revealed identical sequences within the first 17 amino acid residues (band 1: ALKGGACPPRKIVQCLRYEK; band 2: ALKGGACPPRKIVQCLRYE). These sequences corresponded exactly to those predicted from the nucleotide sequence of cloned porcine uterine SLPI cDNA [8]. The purified pUPTI protein migrated as a doublet on a one-dimensional SDS-PAGE gel (Fig. 1B), with estimated molecular weights for the top and bottom bands of 17 000 (intact) and 14 000 (truncated), respectively. The NH2-terminal amino acid sequences of these two proteins (top: VRAGPPAFCREPPYTGP; bottom: VRAGPPAFCREPPYTG) corresponded exactly to those predicted from the nucleotide sequence of cloned porcine UPTI cDNA [15]. The pUPTI protein of 17 kDa was purified from the 14-kDa protein by electroelution from acrylamide gel slices using the Bio-Rad electroeluter (Bio-Rad) according to the manufacturer's instructions and was used in all assays described in the present study.
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Trypsin Inhibition by SLPI and UPTI
The endogenous anti-trypsin activity of pSLPI, pUPTI, and rhSLPI was examined as previously described [15]. All three proteins inhibited trypsin activity at protein amounts within the nanogram-to-microgram range. Porcine SLPI and UPTI had 10% and 20% trypsin inhibitory activities, respectively, at 650 ng, and these increased to 40% and 70%, respectively, at 6.5 µg. Recombinant hSLPI, at 200 and 400 ng, had 20% and 40% trypsin inhibitory activities, respectively.
Mitogenic Effects of SLPI and UPTI in Uterine GE Cells
To evaluate the mitogenic potentials of SLPI and UPTI towards uterine epithelial cells, primary cultures of GE cells at 100% confluence were incubated in serum-free medium for 24 h and then treated with increasing concentrations (10–1000 ng/ml) of pSLPI, rhSLPI, and pUPTI for 20 h. The cells were then pulse-labeled with [3H]thymidine for the last 4 h of the incubation period. All three protease inhibitors increased (p < 0.05) DNA synthesis in GE cells over control (untreated) cells at the different concentrations tested (Figs. 2 and 3). SLPI stimulation of [3H]thymidine incorporation into DNA of GE cells was completely abrogated upon preincubation of pSLPI and rhSLPI with anti-pSLPI (IgG fraction; 30 µg/well) and anti-hSLPI (IgG fraction; 15 µg/well) antisera, respectively, for 2 h before treatment (Fig. 2, C and D). Neither antiserum alone had any effect on labeled thymidine incorporation by these cells (data not shown). The lack of available anti-pUPTI antiserum precluded the conduct of similar experiments with UPTI.
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To examine further whether SLPI and UPTI increase uterine epithelial cell DNA synthesis via common signaling pathways, confluent GE cells were incubated with pSLPI (100 ng/ml) and pUPTI (100 ng/ml) alone or in combination in serum-free media. Simultaneous addition of pSLPI and pUPTI to these cells resulted in stimulation of [3H]thymidine incorporation into DNA to a level higher (p < 0.05) than that obtained with either anti-protease alone (Fig. 4).
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Heparin Effects on SLPI- and UPTI-Induced [3H]Thymidine Incorporation
The glycosaminoglycan heparin had been previously demonstrated to bind hSLPI, resulting in the increased rate of inhibition of its substrate elastase [25]. To determine whether interaction with heparin similarly influences the mitogenic potentials of SLPI and UPTI, the effect of added heparin (0.5 µg/ml) on SLPI- and UPTI-induced [3H]thymidine incorporation into GE cellular DNA was examined. All three protease inhibitors increased (p < 0.05) DNA synthesis in GE cells above control values, and the effects of added pSLPI, hSLPI, and pUPTI on these cells were comparable (p > 0.05) (Fig. 5). However, heparin had no effect (p > 0.05) on the level of labeled thymidine incorporated by DNA of these cells, and exhibited only a tendency (p = 0.08) to enhance the individual effects of all three protease inhibitors.
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SLPI and UPTI Transcript Levels in Porcine Endometrium during Pregnancy
Steady-state pSLPI and pUPTI mRNA levels in endometrium as a function of stage of pregnancy and of cell type were evaluated by Northern analysis and semiquantitative RT-PCR. Northern blot analysis showed the presence of SLPI and UPTI transcripts in Day 12 and Day 60 pregnant pig endometrium, with the level of each transcript greater at Day 60 than at Day 12 (Figs. 6A and 7A). Consistent with this, the levels of SLPI mRNA increased (p < 0.05) between Days 12 and 60 and remained high through Day 90 of gestation (Fig. 6B), as measured by RT-PCR. SLPI transcript levels were consistently present and higher (p < 0.05) in endometrial GE, compared to ST, cells (Fig. 6C). The levels of UPTI transcript were readily detected at Day 12, peaked by Day 60, and were diminished by Day 90 of pregnancy (Fig. 7B). Similar to SLPI, a tendency (p = 0.07) for higher levels of UPTI transcript in GE vs. ST cells was observed in the endometrium of early pregnancy (Fig. 7C). In all cases, the levels of ß2-microglobulin transcript [26], the internal control in the PCR reactions, did not change [27].
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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SLPI (also known as antileukoprotease), although widely present in human mucosal secretions [28, 29], was described in the pig initially as a pregnancy-associated uterine protease inhibitor [8]. Subsequent studies indicated that its high level of uterine endometrial expression during pregnancy appears to be limited to mammalian species with noninvasive placentation (e.g., pig, cow, horse). Species exhibiting more intimate contact of the maternal and trophoblast tissues (e.g., rat, mouse) were found to show undetectable endometrial levels of this mRNA and protein [16]. The latter observation implies that uterine SLPI may largely underlie the maintenance of an intact feto-maternal interface in species with epitheliochorial placenta [21], thus primarily acting as an anti-protease within the uterine environment. However, the finding reported here suggests that SLPI may have an additional function during early pregnancy that involves its modulation of uterine glandular epithelial cell growth. This novel activity of SLPI is specific, since it can be abrogated by the co-addition of specific anti-SLPI antiserum and is demonstrated by SLPI isolated from two mammalian species (human and pig). Additionally, the pig and human SLPI proteins were active within the same concentration range (10–1000 ng/ml), eliminating any possibility that minor contaminants in the pig SLPI protein might underlie the observed activity. The latter observation is particularly interesting since human and porcine SLPI exhibit only a 68% similarity in primary structures [8]; this suggests that the conserved core regions within the two proteins, which include cysteine- and proline-rich residues as well as a two-domain structure [8, 30], are sufficient for these proteins' mitogenic potentials.
Interestingly, another uterine-derived serine anti-protease in the pig, namely uterine plasmin/trypsin inhibitor [9, 15], induced [3H]thymidine incorporation into DNA of GE cells, similar to that of SLPI (this study). Indeed, several features of the mitogenic activities of SLPI and UPTI are worth noting. First, their respective activities are achieved at concentrations (i.e., ng/ml) at which classical growth factors have been shown to be effective ([31] and references therein). Second, their near-maximal mitogenic activities are observed at concentrations at which their anti-trypsin activities are less than optimal. Third, their sum activities are higher than that individually. Finally, their activities appear to be modulated, to a limited extent, by heparin. Taken together, these data indicate that the mitogenic potentials of SLPI and UPTI probably involve mechanisms distinct from their anti-protease functions. TIMPs have been shown to modulate target cell mitogenesis via cell surface receptors [13]; however, our preliminary data indicate lack of binding of labeled pSLPI or hSLPI to monolayer cultures of GE cells or membrane-enriched fractions prepared from these cells (data not shown). This suggests that cell surface receptors probably do not mediate the mitogenic activity of SLPI. Potential mediators might include components of the extracellular matrix such as integrins, which have been shown to directly influence cellular differentiation, survival, and function [32]. In this regard, SLPI has been localized to the extracellular matrix via its association with elastin fibers in the human lung [33].
The functional domain(s) involved in the biological activity of SLPI can only be speculated on at the present time. The observation that alkylated TIMPs had no anti-metalloprotease activity, while maintaining significant cell-proliferating activity [13], indicate that distinct domains are responsible for the mitogenic and anti-protease activities of these protease inhibitors. The anti-trypsin activity of SLPI has been localized to its carboxy-terminal domain [34, 35], while the positively charged amino acid residues that bind heparin are dispersed in clusters throughout the molecule [36]. Since heparin appears to enhance SLPI-induction of DNA synthesis, albeit to a much lesser extent that its inhibitory activity [24, 37], the specific domains involved in these distinct functions of SLPI cannot be assigned with certainty.
Finally, the biological rationale for the mitogenic activity of SLPI and UPTI towards uterine GE cells remains unclear. The three major cell types of the uterine endometrium (GE, LE, and ST) synthesize a diverse array of classical growth factors, which together would appear to be sufficient to provide an environment optimal for growth [38]. Our previous finding that a conceptus secretory product, transforming growth factor , induces endometrial expression of the pSLPI gene [14], whose product is now demonstrated to exhibit endogenous mitogenic activity, suggests that a period critical for periimplantation may require a rapid but sustained local amplification of growth. At later pregnancy stages (e.g., mid-pregnancy), when SLPI and UTPI synthesis are maximal (this study; [8, 15]) and, as in the case for SLPI, the protein is transported to the fetal circulation [21], the possibility that these serine protease inhibitors function less as mitogenic agents and more as anti-proteases is likely. Alternatively, SLPI may have additional functions distinct from the above. A novel role for SLPI in the inhibition of matrix metalloproteinase production by monocytes [39] suggests its anti-inflammatory action, independent of its serine protease substrates.
In conclusion, our results provide evidence for a novel autocrine function for SLPI and UPTI in early pregnancy events. These data, taken in light of similar demonstrated activities for members of another protease inhibitor family, namely TIMPs 1 and 2, suggest that control of cell growth may constitute a general function for protease inhibitors in mammals. The elucidation of the mechanisms that underlie this activity distinct from their respective anti-protease functions may provide a clear understanding of the signaling mechanisms and novel strategies for ensuring successful implantation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 352 392 7652; simmen{at}animal.ufl.edu
Accepted: March 9, 1999.
Received: November 12, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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