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Originally published In Press as doi:10.1074/jbc.M203503200 on May 22, 2002

J. Biol. Chem., Vol. 277, Issue 33, 29999-30009, August 16, 2002
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Secretory Leukocyte Protease Inhibitor Mediates Proliferation of Human Endometrial Epithelial Cells by Positive and Negative Regulation of Growth-associated Genes*

Daying Zhang, Rosalia C. M. Simmen, Frank J. Michel, Ge Zhao, Dustin Vale-Cruz, and Frank A. SimmenDagger

From the Interdisciplinary Concentration in Animal Molecular & Cell Biology and the Department of Animal Sciences, University of Florida, Gainesville, Florida 32611-0910

Received for publication, April 11, 2002, and in revised form, May 7, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Secretory leukocyte protease inhibitor (SLPI) inhibits chymotrypsin, trypsin, elastase, and cathepsin G. This protein also exhibits proliferative effects, although little is known about the molecular mechanisms underlying this activity. We have generated SLPI-ablated epithelial sublines by stably transfecting the Ishikawa human endometrial cell line with an antisense human SLPI RNA expression vector. We demonstrate a positive correlation between cellular SLPI production and proliferation. We further show that Ishikawa sublines expressing low to undetectable SLPI have correspondingly increased and decreased expression, respectively, of transforming growth factor-beta 1 and cyclin D1 genes, relative to parental cells. SLPI selectively increased cyclin D1 gene expression, with the effect occurring in part at the level of promoter activity. Cellular SLPI levels negatively influenced the anti-proliferative and pro-apoptotic insulin-like growth factor-binding protein-3 expression. We also identified lysyl oxidase, a phenotypic inhibitor of the ras oncogenic pathway and a tumor suppressor, as SLPI-repressed gene, whose expression is up-regulated by transforming growth factor-beta 1. Our results suggest that SLPI acts at the node(s) of at least three major interacting growth inhibitory pathways. Because expression of SLPI is generally high in epithelial cells exhibiting abnormal proliferation such as in carcinomas, SLPI may define a novel pathway by which cellular growth is modulated.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Secretory leukocyte protease inhibitor (SLPI),1 also known as anti-leukoprotease, human mucus proteinase inhibitor, and human seminal plasma inhibitor, is a 12-kDa member of the chelonianin class of serine protease inhibitors, which also includes SKALP/elafin (1, 2). SLPI is composed of two homologous cysteine-rich domains. The carboxyl-terminal domain manifests inhibitory activities against chymotrypsin, trypsin, granulocyte and pancreatic elastases, cathepsin G, and mast cell chymase (3, 4). SLPI is expressed primarily in secretory/glandular epithelial cells of a variety of human tissues including the bronchi, parotid glands, small and large intestines, skin, breast, pancreas, male and female genital tracts, and kidney (5-14). Its relative absence in serum, except in certain pathological conditions (15, 16), and its localization to extracellular matrix and subcellular sites not readily accessible to larger molecular weight protease inhibitors such as alpha 1-antitrypsin (17), suggests a primarily autocrine/paracrine mode of action. In addition to its anti-protease activity, SLPI has anti-inflammatory, anti-bacterial, anti-fungal, and anti-retroviral (human immunodeficiency virus) activities that appear to reside in its amino-terminal cysteine-rich domain (18-23). Furthermore, SLPI has been shown to regulate intracellular enzyme synthesis, suppress matrix metalloproteinase production and activity, mediate normal wound healing, prevent scar formation, and augment fertility (24-27). The higher uterine endometrial expression of SLPI during pregnancy in species with the epitheliochorial, as opposed to the hemochorial type of placentation, suggests an important role for this protein in the maintenance of an intact utero-placental interface (28). Its relevance to the regulation of pregnancy-associated events is further bolstered by the recent findings of elevated SLPI expression in endometriotic tissues and corresponding peritoneal fluids (29), in amniotic fluids during the onset of labor (30), and in human fetal membranes and cervical mucus in normal pregnancy (13).

A possible role for SLPI in the control of cell proliferation was initially gleaned from the high levels of SLPI found in epithelial carcinomas (31, 32). Subsequently, SLPI was shown to support the growth of human hematapoietic progenitor cells in serum-free medium in vitro (33), and to increase the production of hepatocyte growth factor in human lung fibroblasts (34). Our own studies demonstrated that SLPI, added at levels comparable with those found in uterine luminal fluids in vivo, elicited an increase in DNA synthesis in primary cultures of glandular epithelial cells isolated from pregnant pig uterine endometrium (35). Moreover, clonal lines of the human endometrial epithelial cell line Hec-1-A overexpressing the transcription factor BTEB1 (Basic Transcription Element-Binding protein 1) and, consequently, exhibiting higher proliferative potential as well as increased expression of a number of cell cycle-associated genes, have augmented SLPI mRNA and protein levels, relative to more slowly growing clonal lines with lower BTEB1 expression (36). Although the increased proliferative responsiveness to serum of the higher expressing BTEB1 lines has now been partially elucidated by the identification of BTEB1 gene targets and by the demonstration of BTEB1 transactivation of promoters for a number of growth-associated genes (37), the molecular mechanism underlying SLPI regulation of cell proliferation remains unresolved.

In the present study, we used the well differentiated human endometrial adenocarcinoma cell line Ishikawa (38) to establish sublines that stably express antisense SLPI RNA. By using these derived clonal lines, we have directly linked cellular SLPI production to proliferation. We also show that SLPI specifically increases cyclin D1 gene expression via the transcriptional activation of its promoter, and we identify LOX as an SLPI negatively regulated gene using the methodology of differential display-reverse transcriptase-PCR. Finally, we demonstrate an inverse relationship between expression of SLPI and mRNA levels of two potent epithelial growth inhibitors, namely TGF-beta 1 and IGFBP-3. These results suggest a novel function for SLPI in epithelial cell proliferation that is mechanistically distinct from its general protease inhibitory and antibiotic activity.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Reagents were obtained as follows. Restriction enzymes and Taq DNA polymerase were from Roche Molecular Biochemicals; nick-translation kit was from Amersham Biosciences; [alpha -32P]dCTP (3000 Ci/mmol) and Biotrans nylon membranes (0.2 µm) were from ICN Radiochemicals (Irvine, CA); cell culture media were from Invitrogen; anti-cyclin D1 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA); anti-PCNA (proliferative cell nuclear antigen) antibody was from Roche Molecular Biochemicals; and pIND mammalian expression vector was from Invitrogen. All molecular biology grade chemicals and solvents, when not specified, were purchased from Fisher.

Cell Culture and Treatments-- The human endometrial carcinoma cell line Ishikawa (courtesy of Dr. Bruce Lessey, University of North Carolina, Chapel Hill) was routinely cultured in minimal essential medium (MEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and antibiotic/antimycotic solution at 37 °C in an atmosphere of 95% air, 5% CO2. Cells were grown in the same medium until they reached 90% confluence, at which time the medium was changed to serum-free. Twenty four hours later, cells received either recombinant human SLPI (100 ng/ml; R & D Systems, Inc., Minneapolis, MN) twice at 24-h intervals, or TGF-beta 1 (10 ng/ml; R & D Systems, Inc., Minneapolis, MN) was added once. Cells were then collected 24 h after the last treatment. Control cells received serum-free medium lacking the treatment and were incubated for the same length of time.

Generation of Stably Transfected Cell Lines-- The full-length human SLPI cDNA (399 bp) was amplified by reverse transcriptase (RT)-PCR from Ishikawa cell RNA using forward (5'-ATGAAGTCCAGCGGCCTCTTC-3') and reverse (5'-TCAAGCTTTCACAGGGGAAA-3') primers that were synthesized based on the sequence for human SLPI mRNA (GenBankTM accession number AF114471). This fragment was cloned into the TOPOTM TA cloning vector (Invitrogen), cleaved with EcoRI, and subcloned into pIND mammalian expression vector (Invitrogen). The sense and antisense orientations of the cloned fragments were determined by digestion with EcoRI and HindIII. Plasmid DNAs were prepared using the Qiagen Plasmid Maxi Kit (Qiagen, Chatsworth, CA), sequenced to confirm the fidelity of the constructs (Interdisciplinary Center for Biotechnology Research, University of Florida), and used to transfect Ishikawa cells using LipofectAMINE (Invitrogen), following the manufacturer's instructions. Transfected cells were cultured for 4-6 weeks in serum (10% FBS)-containing MEM supplemented with G418 (1 mg/ml; Invitrogen), and surviving colonies were selected using cloning cylinders. Individual colonies were expanded in the same medium with added G418 (1 mg/ml). Clonal lines were verified to express the antisense SLPI mRNA by RT-PCR, utilizing cDNAs synthesized from the total RNA of selected cell lines and primers (forward, 5'-CTCTGAATACTTTCAACAAGTTAC-3'; reverse, 5'-CTGTGGAAGGCTCTGGAAAG-3') located within the pIND vector 5'-untranslated region and the antisense SLPI cDNA sequence, respectively. The presence of the amplified PCR product (451 bp) was evaluated by electrophoresis in a 1% agarose gel followed by ethidium bromide staining.

Cell Number and DNA Synthesis-- Clonal lines stably expressing the empty vector alone (13pIND, control) or the antisense SLPI construct (8As) were seeded into 6-well tissue culture dishes at the same density (1.2 × 105 cells/well) in serum-containing medium 2 days before the start of the cell count (designated as day -2). Cells were allowed to adhere to the plates for 24 h and were induced to growth arrest by changing the growth medium to serum-free. After 24 h, the cells received fresh medium containing 10% FBS. Every other day for a total of 8 days, the cells were counted under a microscope using a hemocytometer. The experiment was repeated three times, with each experiment carried out in triplicate wells.

To monitor cellular DNA synthesis, cells were seeded at a density of 6 × 105 cells/well in 6-well, tissue culture dishes. At confluence, cells were incubated in serum-free medium for 24 h and then transferred to serum-containing medium for another 24 h. At 4 h before the end of the incubation period, cells were pulse-labeled with [3H]thymidine (1 µCi/well). The processing of cells to quantify incorporated label followed previously described protocols (39).

RNA Isolation and Northern Blot Analysis-- Total cellular RNA (30 µg), prepared by the Trizol method, was electrophoresed in 1% agarose/formaldehyde gels in 1× MOPS buffer and blotted onto BioTrans nylon membranes (ICN Biotech, Irvine, CA) by downward capillary transfer using the TurboBlotting system (Schleicher & Schuell). RNA was immobilized by UV cross-linking for 90 s followed by baking in an 80 °C oven for 25 min. Blots were pre-hybridized in ULTRAhybTM (Ambion, Austin, TX) at 42 °C for 2 h. Hybridization was carried out overnight in the same buffer containing 32P probes prepared by nick translation (Amersham Biosciences). The DNA fragments used for probe preparation included the following: (a) the human SLPI RT-PCR product (399 bp); (b) the partial cDNA fragments for the cell cycle components cyclin D1 (482 bp), TGF-beta 1 (326 bp), Cdk4 (399 bp), PCNA (520 bp), and p21 (283 bp) (40) generated by RT-PCR, using cDNAs from Ishikawa total RNA as template and primer sets that were synthesized based on the reported human cDNA sequences (GenBankTM); and (c) the EcoRI inserts of the plasmid DNAs obtained from TOPO cloning of differentially expressed cDNAs identified by mRNA differential display (below). The membranes were sequentially washed twice in 2× saline sodium citrate (SSC), 0.1% SDS and in 0.1× SSC, 0.1% SDS at 42 °C for 15 min each time. The blots were exposed to x-ray films using intensifying screens, and resultant hybridization signals were quantified using the Alpha Imager 2000 Documentation and Analysis System (Alpha Innotech Co., San Leandro, CA). The probe was removed after each hybridization reaction by washing the filter twice for 20 min each in 1% SDS solution heated at 100 °C. A partial cDNA fragment (971 bp) corresponding to human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) coding sequence (41) was labeled by nick translation and used to normalize for loading differences among samples.

mRNA Differential Display-- Analysis of differentially expressed mRNAs among clonal sublines utilized the methodology of differential display-reverse transcriptase (ddRT)-PCR performed as described previously in publications from this laboratory (42, 43). DNA-free total RNAs (2 µg) isolated from the parental Ishikawa cell line and the clonal lines 13pIND, 11As, and 8As were each subjected to reverse transcription using anchored 3'-oligo(dT) primer sets (HIEROGLYPHTM, GENOMYX Corp., Foster City, CA). One-tenth of the reverse transcription reaction (20 µl) was used for PCR amplification in a reaction (20 µl total volume) containing 1 unit of AmpliTaq DNA polymerase (PerkinElmer Life Sciences), 400 µM of each dNTP, 2.5 µCi of [alpha -33P]dATP, and two primers: 4 µM of one of T12 oligonucleotides and 4 µM of an arbitrary decamer, M13r-ARP1 (5'-CGACTCCAAG-3') or M13r-ARP2 (5'-GCTAGCATGG-3'). The PCR products from different primer combinations for each of the four cell lines examined were generated following amplification conditions described previously (43), separated in non-denaturing polyacrylamide (4.5%) sequencing gels, and visualized by autoradiography. A number of bands exhibiting differential expression were excised from the dried gels and re-amplified by PCR using the appropriate primers. These fragments were used as probes in Northern blots and were cloned into TOPOTM TA vector for nucleotide sequencing when established to be differentially expressed. The identities of the isolated clones were determined by homology comparisons with sequences in GenBankTM (www.ncbi.nlm.nih.gov/BLAST/).

Transient Transfection and Luciferase Assays-- The full-length human SLPI coding region was released from the plasmid DNA pT7T3D-hSLPI (IMAGE: 796471, IncyteGenomics Inc., Palo Alto, CA) by digestion with EcoRI and NotI, and subsequently ligated into the pcDNA3 vector (Invitrogen) that was previously linearized with the same enzymes. The cyclin D1 reporter construct (-1745CD1LUC in pcDNA3.1/Zeo+ vector) contained the promoter and 5'-regulatory sequences of the human cyclin D1 gene (44) and was generously provided by Drs. Chris Albanese and Richard G. Pestell (Albert Einstein College of Medicine, New York).

For transient transfections, the antisense subline 8As cells were seeded at a density of 6 × 105 cells/well in serum-containing medium. Twenty four h thereafter, cells were incubated with reporter construct (-1745CD1LUC; 5 µg/well) and SLPI expression construct (pcDNA3-hSLPI; 0.5, 1 or 2 µg/well) or corresponding empty vector (pcDNA3) in the presence of hexadimethrine bromide (Polybrene, 5 µl/well; Sigma), for 4-6 h in Hanks' balanced salt solution (HBSS, pH 7.4). Cells were washed with HBSS once, treated with 25% dimethyl sulfoxide (Me2SO) in HBSS for 4 min to increase cell permeability, and then washed twice with the same buffer prior to their incubation in serum-containing MEM for an additional 48 h. Luciferase activity (measured as relative light units) in cell lysates prepared using single-strength lysis buffer was determined using the Promega luciferase assay system. Three independent transfection experiments were performed, with each experiment carried out in triplicate. Results were normalized to the protein content of each sample and are presented as least squares means ± S.E. Protein concentration of extracts was determined by the Bradford method (45).

In studies where the incorporation of labeled thymidine was measured after transient transfection with the SLPI expression construct or empty vector, transfections of the clonal line 8As were carried out using LipofectAMINE, following protocols described previously (46).

Immunofluorescence and Western Blots-- The clonal 13pIND and 8As lines were seeded on immunofluorescence chamber slides (Nalge Nunc International Corp., Naperville, IL) at a density of 1 × 105 cells. The following day, cells were washed with phosphate-buffered saline (PBS), incubated with 4% paraformaldehyde for 10 min, rinsed with PBS three more times, and then incubated with 0.1% Triton X-100 in PBS for 10 min to increase nuclear permeability of antibody. Incubation with rabbit anti-human cyclin D1 polyclonal antibody (final concentration of 4 µg/ml; R & D Systems) or normal rabbit serum IgG (negative control; 4 µg/ml) diluted in 3% bovine serum albumin/PBS was carried out in a humidified box for 1 h. Detection was performed with anti-rabbit fluorescein isothiocyanate-conjugated secondary antibodies (Sigma; 1:200 dilution) for 45 min. Cells were photographed using the Zeiss Axioplan 2 Fluorescence Microscope (Carl Zeiss Microimaging, Inc., Thornwood, NY).

Standard Western blot techniques were used to determine the levels of PCNA in cell nuclear extracts prepared following procedures described previously (47), using anti-PCNA monoclonal antibody (1:2000 dilution). SLPI protein in conditioned medium was detected using rabbit polyclonal anti-human SLPI antibody that was generously provided by Dr. P. S. Hiemstra (Leiden University, The Netherlands).

Ligand Blot Analysis-- Confluent 13pIND and 8As clonal lines were incubated in serum-free medium, and conditioned medium was collected 24 h later. Conditioned medium was also collected from confluent 11As and 13pIND clonal lines incubated for 24, 48, and 72 h in serum-free medium. The samples were concentrated, and aliquots corresponding to 30 µg of protein for each sample were electrophoresed in 10% SDS-PAGE. Standard ligand blot procedures using 125I-IGF-I as probe (5 × 106 total counts per membrane) were followed (48).

Statistical Analysis-- The intensities of hybridization signals obtained from Northern blots were normalized to those for the control probe, GAPDH, and analyzed for differences using one-way analysis of variance. The transfection data were normalized to cellular protein content and analyzed by two-way analysis of variance. Results are presented as least squares means ± S.E. Differences with p values <0.05 were considered statistically significant.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of SLPI Antisense Clonal Lines-- Clonal lines derived by stable transfection of Ishikawa cells with an expression construct containing the entire coding region of human SLPI in antisense orientation were confirmed for expression of this construct by RT-PCR using a primer set located within the pIND vector 5'-untranslated region (forward) and the SLPI cDNA sequence (reverse), respectively (Fig. 1A). The relative expression of the antisense construct for each clonal line was determined from ethidium bromide-stained agarose gels containing the generated 451-bp RT-PCR fragment (Fig. 1B) and was verified by Northern and Western blot analyses of corresponding cellular RNA and protein, respectively, for SLPI (Fig. 1C). The level of abundance of the 451-bp fragment in the Antisense (As) lines 8, 11, and 18 (8 > 11 > 18) was inversely correlated with their expression of endogenous SLPI mRNA and protein (8 < 11 < 18). As expected, control lines (3pIND, 13pIND) stably expressing empty vector had SLPI mRNA and protein levels that were indistinguishable from those of parental (untransfected) cells (Fig. 1C). Because the functional consequence of SLPI ablation was the major focus of the present study, the sublines that exhibited the most dramatic differences in SLPI gene expression, namely 8As, 11As, and the control 13pIND, were used for much of the subsequent analysis.


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  		Fig. 1.   Expression of SLPI in stably transfected human Ishikawa clonal cell lines. A, schematic representation of the entire coding region of human SLPI (hSLPI) cDNA inserted in antisense orientation in the pIND expression plasmid, and the locations of the forward and reverse primers used in RT-PCR to confirm antisense RNA expression from the chimeric DNA. B, analysis of chimeric antisense (As) SLPI RNA expression by RT-PCR. cDNAs were synthesized from total RNA isolated from 8As, 11As, and 18As sublines and used as templates in PCRs using primers described above and in the text. The expected PCR fragment of 451 bp is demonstrated. C, Northern and Western blot analysis of SLPI in parental Ishikawa cells and derived clonal lines. Total RNA (30 µg/lane) isolated from the indicated cell lines was probed with alpha -32P-hSLPI (399 bp) or [alpha -32P]hGAPDH (971 bp) cDNA fragments. The conditioned medium from the indicated cell lines was concentrated and loaded (10 µg of protein/lane) onto 10% SDS-PAGE. SLPI was detected using a rabbit anti-human SLPI polyclonal antibody and the ECL detection system.

SLPI Is an Epithelial Cell Growth Factor-- The effect of SLPI on epithelial cell number was evaluated for the two clonal lines that had undetectable (8As) and high (13pIND) levels of SLPI expression, respectively. Cells were seeded at the same density, transferred to serum-free medium to synchronize cell cycle stage, and then transferred back to serum-containing medium for the duration of the study. As shown in Fig. 2A, cell growth rate was higher in the 13pIND line than in the 8As line at all time points examined; indeed, 6 days after plating, the cell density observed for the control line was nearly 6-fold higher than for the SLPI antisense line. Consistent with this, cellular DNA synthesis, quantified by labeled thymidine incorporation, was higher in 13pIND than 8As line (Fig. 2B). The clonal line 11As shown earlier to express SLPI at levels between those of control and 8As lines (Fig. 1C) exhibited DNA synthetic rates that were also mid-way between the two lines. These results indicate a positive correlation between SLPI expression and epithelial cell proliferation.


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  		Fig. 2.   Distinct growth parameters of Ishikawa sublines are dependent on cellular SLPI content. A, control (13pIND) and antisense SLPI (8As) sublines were seeded at a density of 1.2 × 105 cells/well in serum-containing medium (-2 day). Cells were allowed to adhere to the plates for 24 h, and medium was then changed to serum-free. After an additional 24 h, cells received fresh medium containing 10% FBS (day 0). Every other day for a total of 8 days, the cells were counted. B, DNA synthesis in control (13pIND) and antisense SLPI (11As and 8As) sublines. Cells were seeded at the same density (6 × 105/well) and were allowed to attach to the dishes. After serum starvation for 24 h, cells were changed to serum-containing medium for another 24 h. Cells were pulse-labeled with [3H]thymidine for 4 h. C, DNA synthesis in the 8As subline in the presence or absence of transfected human SLPI expression vector. Cells were transiently transfected with the SLPI expression construct (pcDNA3-hSLPI) or empty vector (pcDNA3), and the assay for [3H]thymidine incorporation was carried out 24 h later. Data presented in A-C (mean ± S.E.) are from three independent experiments, with each experiment carried out in triplicate.

To examine further this relationship, the 8As clonal line that had undetectable expression of SLPI was transiently transfected with a human "sense" SLPI expression construct (pcDNA3-hSLPI) to override the antisense effect (empty pcDNA3 vector was transfected in parallel as control), and thymidine incorporation by transfected cells was measured. Cellular DNA synthesis in the 8As clonal line that was transfected with pcDNA3-hSLPI was greater than in cells transfected with empty vector (Fig. 2C). These results further confirm that SLPI promotes endometrial epithelial cell proliferation.

Expression Levels of Cyclin D1 and TGF-beta 1 Are Modulated by SLPI-- To determine whether the modulation by SLPI of epithelial cell proliferation involves specific changes in cell cycle- and growth-associated gene expression, the mRNA levels for a number of cell cycle-related proteins were monitored in several clonal As-SLPI lines and, for comparison, in the parental Ishikawa cell line and the control pIND sublines. As shown in Fig. 3, the levels of cyclin D1 and of TGF-beta 1 mRNAs were positively and negatively correlated, respectively, with those of SLPI. When normalized to the expression of the control GAPDH gene, which was unaffected by cellular SLPI expression, these positive (cyclin D1) and inverse (TGF-beta 1) relationships with SLPI were more evident (data not shown). In particular, the 8As clonal line, which had undetectable SLPI gene expression, had the lowest and highest levels, respectively, of cyclin D1 and TGF-beta 1 mRNAs. By contrast, the expression levels of cdk4, PCNA, and p21 genes were relatively unchanged in all clonal lines, irrespective of SLPI gene expression levels. Immunofluorescence with a specific antibody against human cyclin D1 was used to examine for differences in the levels of this protein in 8As and control (13pIND) sublines. Consistent with the differences noted in cyclin D1 mRNA levels, the nuclear abundance of cyclin D1 in 8As was less than that observed for 13pIND (Fig. 4). In particular, almost all 13pIND cells exhibited staining, whereas that for 8As was more irregular and spotty. The specificity of the antigen-antibody reaction was verified by virtue of the undetectable nuclear staining observed in both lines when control rabbit IgG was used in place of the test antibody. Furthermore, the inductive effect of SLPI was selective for cyclin D1 because the levels of PCNA protein were not different in nuclear extracts prepared from 8As and 13pIND sublines (data not shown), in agreement with the corresponding mRNA data (Fig. 3).


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  		Fig. 3.   Expression of cell cycle-associated genes in parental Ishikawa and derived clonal cell lines with distinct SLPI expression. Cells were grown to confluence in serum-containing medium and then used for RNA isolation. Total RNA (30 µg/lane) was analyzed for mRNA content for the indicated genes using alpha -32P-labeled cDNA fragments as probes.


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  		Fig. 4.   Abundance of cyclin D1 protein in control (13pIND) and SLPI-deficient (8As) sublines. Cells were seeded at a density of 1 × 105/well on immunofluorescence chamber slides. The following day, cells were incubated with rabbit anti-human cyclin D1 polyclonal IgG (4 µg/ml) (bottom panels) or normal rabbit serum IgG (4 µg/ml) (top panels), followed by fluorescein isothiocyanate-conjugated secondary antibodies. Photographs were taken using the Zeiss Axioplan 2 fluorescence microscope.

SLPI Modulates Expression of Growth-associated Genes-- To explore further the mechanism(s) behind the SLPI regulation of epithelial cell proliferation, we used the methodology of mRNA differential display in an attempt to isolate other differentially expressed growth-related genes. From this analysis, we identified an mRNA of 4.5 kb in length whose expression was lower in cells expressing more SLPI (Fig. 5A) to encode LOX, a key enzyme involved in the control of collagen and elastin cross-linking (49, 50), and which has been shown to act as a tumor suppressor by virtue of its ability to inhibit the protooncogene p21ras pathway (51-53). Another mRNA (A-10, 1.3 kb in length) whose expression was lower in the 8As control line was also isolated. However, the identity of this mRNA is presently unknown because its sequence does not correspond to any of those currently deposited in the GenBankTM. The Northern analysis for a third cDNA (A-13) is also shown (Fig. 5A); this transcript was not found to be differentially expressed and served as a control probe for RNA loading and integrity.


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  		Fig. 5.   Differential expression of growth-associated genes in stably transfected Ishikawa sublines. A, total cellular RNA isolated from parental Ishikawa cells (Normal) and clonal lines stably transfected with empty vector (13pIND) or antisense SLPI expression construct (11As and 8As) were subjected to mRNA differential display RT-PCR as described under "Experimental Procedures." DNA fragments representing potential differentially expressed transcripts were used as probes in Northern blots. Nucleotide sequence analysis identified the 4.5-kb transcript as that for lysyl oxidase (LOX). The ddRT-PCR product was derived from the 3'-untranslated region of exon 7 of this gene. The identity of the 1.3-kb transcript for clone A-10 is currently unknown. Clone A-13 is a control probe from ddRT-PCR and is not differentially expressed. B, confluent 13pIND and 8As clonal lines were incubated in serum-free medium, and conditioned medium was collected 24 h later. Conditioned medium was also collected from 11As and 13pIND clonal lines incubated for 24, 48, and 72 h in serum-free medium. The samples (30 µg of protein/lane) were concentrated and subsequently analyzed for presence of IGF-binding proteins using 125I-IGF-I as the probe for ligand blots. The two radioactive bands of ~51 kDa represent the known glycosylation variants of IGFBP-3, whereas the lower band of 36 kDa is IGFBP-2.

We also employed the IGF ligand blot procedure to evaluate whether changes in the levels of IGF-binding proteins were correlated with cellular SLPI, because Ishikawa cells are autocrine targets of IGF-I (54). The antisense SLPI lines (8As and 11As) and the control line 13pIND demonstrated the presence in the conditioned medium of two major IGF-binding proteins, namely IGFBP-2 and IGFBP-3. However, whereas the levels of IGFBP-2 did not change among the three sublines, those for IGFBP-3 were increased in the antisense lines (Fig. 5B). Moreover, the levels of IGFBP-3 appeared to be inversely correlated with those of SLPI, because 11As (higher SLPI gene expression) had less secreted IGFBP-3 than did 8As.

We next examined if addition of SLPI to the medium of antisense lines could alter their patterns of gene expression to mimic that of the control (pIND) line. Changes in gene expression in sublines to which had been added recombinant human (rh) SLPI twice over a 48-h interval were measured by Northern blots. Consistent with our earlier observations (Fig. 3 and Fig. 5), the basal expression levels of the TGF-beta 1, IGFBP-3, and LOX genes were negatively correlated with endogenous SLPI expression, whereas that of cyclin D1 was positively correlated (Fig. 6A). However, addition of rhSLPI had an effect only on cyclin D1 mRNA levels, which increased by ~3-fold relative to basal levels, and only in the 8As line. By contrast, expression of the other genes that were similarly evaluated in all sublines were unaffected by rhSLPI addition.


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  		Fig. 6.   Gene expression by control and SLPI-deficient clonal lines upon SLPI administration. A, control (pIND) and antisense SLPI (11As and 8As) cell lines were grown to confluence and then transferred to serum-free medium. After 24 h, cells received either human recombinant SLPI (100 ng/ml) or buffer alone twice at 24-h intervals. Cells were collected 24 h after the last treatment. Total RNA was isolated from cells and analyzed by Northern blot using alpha -32P-labeled inserts for SLPI, cyclin D1, TGF-beta 1, IGFBP-3, and LOX cDNAs as probes. The bar graphs (least square means ± S.E.) represent the hybridization intensities of each transcript after normalizing to that of GAPDH. B, cells at 70% confluence were transiently transfected with either empty vector (pcDNA3) or hSLPI expression construct (pcDNA3-hSLPI) using LipofectAMINE, following procedures described in the text. Northern blot analysis was carried out on total cellular RNAs isolated from these cells, using the various probes listed in A. Only the results of experiments using SLPI and cyclin D1 as hybridization probes are shown here. Each experiment was repeated three times.

Transient transfection of the sense human SLPI expression vector into the 8As line was used to assess the consequences of increasing intracellular and extracellular levels of SLPI on the expression of cyclin D1 as well as other genes. The expression vector significantly increased, albeit modestly, the levels of cyclin D1 (Fig. 6B), but not of TGF-beta 1, IGFBP-3 and LOX, mRNAs (data not shown) relative to empty vector in this subline. Basal SLPI mRNA levels were increased by at least 4-fold with transfected SLPI expression vector relative to empty vector (Fig. 6B). Together, these results suggest that SLPI has a direct regulatory role on cyclin D1 gene expression, whereas its effects on the expression of the other genes may be mediated by indirect mechanisms.

SLPI Affects Cyclin D1 Promoter Activity-- The mechanism behind SLPI regulation of cyclin D1 gene expression was examined by evaluating whether SLPI modulates cyclin D1 promoter activity. The 8As line was transiently co-transfected with the cyclin D1-Luc reporter construct and human SLPI expression vector or corresponding empty vector, and luciferase activity was measured in resultant cell lysates. SLPI had a dose-dependent effect on cyclin D1 promoter activity, with no effect of the added expression vector at 0.5 µg, a modest inductive effect at 1.0 µg, and a dramatic inhibitory effect at 2 µg (Fig. 7). This suggests that SLPI regulates cyclin D1 gene expression at the level of transcription, although the direction of regulation (transactivation or transrepression) appeared to be dose-dependent.


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  		Fig. 7.   Effect of SLPI on cyclin D1 promoter activity. The 8As subline was seeded at a density of 6 × 105 cells/well. Twenty four h later, cells were transiently co-transfected with -1745CD1LUC reporter construct (5 µg/well) and an SLPI expression vector (pcDNA3-hSLPI; 0.5, 1, or 2 µg/well) or the corresponding empty vector pcDNA3, and luciferase assay on cell lysates was carried 48 h later. Three independent transfection experiments were carried out, with each experiment performed in triplicate. Results were normalized to the protein content for each sample and are presented as least square means ± S.E.

TGF-beta 1 Opposes SLPI Effects on Gene Expression-- The lack of a direct effect of SLPI, whether added exogenously or produced intracellularly via transfection, on the expression of IGFBP-3 and LOX genes, despite the observed strong negative correlation of their expression with that of SLPI in antisense lines, suggests indirect mode(s) of SLPI control. Because the expression levels of SLPI and TGF-beta 1 genes are inversely correlated in the Ishikawa sublines described here, we examined whether TGF-beta 1 had a direct effect on IGFBP-3, LOX, and cyclin D1 gene expression, respectively. In these studies, parental Ishikawa cells were incubated in serum-free medium with or without added rhTGF-beta 1 for 24 h, and total cellular RNA from treated and control cells was analyzed for expression of these genes (Fig. 8) by Northern blotting. Relative to untreated cells, TGF-beta 1 decreased SLPI (~1.5-fold) and IGFBP-3 (~2.5-fold), increased LOX (~3-fold) and its own (~2.5-fold), and had no effect on cyclin D1 and GAPDH mRNA levels. These findings suggest a regulatory loop between SLPI and TGF-beta 1, whereby induction of TGF-beta 1 gene expression is a direct or indirect consequence of diminished SLPI expression, and this can result in increased levels of LOX.


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  		Fig. 8.   Effects of TGF-beta 1 on Ishikawa cell gene expression. Parental Ishikawa cells grown in serum-containing medium until 90% confluent were transferred to serum-free medium for 24 h, prior to treatment with human TGF-beta 1 (10 ng/ml). Twenty four h later, cells were collected, and the total RNA was isolated and analyzed for SLPI, TGF-beta 1, cyclin D1, LOX, IGFBP-3, and GAPDH gene expression. The Northern blot data for duplicate cultures are shown.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study identifies a novel mechanism by which SLPI, a multisubstrate anti-protease member of the Serpin family, functions as an epithelial cell growth factor. Several lines of evidence presented herein suggest that the growth-regulatory role of SLPI, as measured by its ability to increase cell number as well as DNA synthesis, is mediated by its activation as well as its inhibition of distinct cell cycle and growth-associated gene expression. In particular, the following findings are consistent with a regulatory role for SLPI in growth-signaling pathways: 1) SLPI increased cyclin D1 gene expression at the level of the promoter activity of this gene; 2) cellular SLPI levels are inversely correlated with those of TGF-beta 1 mRNA and IGFBP-3 mRNA and secreted protein; 3) the expression of the gene encoding LOX, a phenotypic inhibitor of the oncogenic ras pathway (52, 53), is markedly up-regulated in sublines with undetectable SLPI; and 4) TGF-beta 1 inhibits SLPI gene expression. The diverse actions of SLPI highlighted here expand the repertoire of SLPI-regulated genes and encoded products, which previously have included the nuclear factor (NF)-kappa B inhibitor Ikappa Bbeta (55) and prostaglandin H2 synthase-2 (24), to those linked to growth control, and implicate distinct mechanisms employed by SLPI in modulating cell function and phenotype.

A most intriguing finding is the identification of cyclin D1 as a downstream target of SLPI. The ability of SLPI to induce cyclin D1 expression occurred at the levels of its mRNA, protein, and promoter activity; is highly specific as evident from the lack of comparable effects on the expression levels of other G1 phase-associated genes such as cdk4, PCNA, and p21; and was observed irrespective of the mode of SLPI delivery (intracellular or extracellular). A supportive role for SLPI in growth processes had been observed previously (34, 35) in epithelial and fibroblastic cell types, although the molecular mechanism underlying this function remained unclear. Members of the tissue inhibitor of metalloproteinase (TIMP) family, specifically TIMP-1 and TIMP-2, exhibit growth-promoting activities, which appear to be mediated by signal transduction pathways independent of their protease inhibitor functions and involve cAMP (56-58). The immediate and direct route for SLPI to modulate cell cycle progression via regulation of cyclin D1 expression is consistent with a mechanistically distinct pathway from that utilized for its protease inhibitory function, although this contrasts with previous findings of the requirement for the trypsin inhibitory activity of this protein in the up-regulation of hepatocyte growth factor gene expression (34). Interestingly, SLPI transactivation of the cyclin D1 gene appears to be dose-dependent and was markedly reversed by a 2-fold increase in the amount of transfected SLPI vector. Although the reason for this is presently unclear, this may be related in part to a previously reported (55) activity of SLPI to inhibit the nuclear accumulation of NF-kappa B via its maintenance of the expression of Ikappa Bbeta , a crucial step in NF-kappa B activation. Because NF-kappa B is a transactivator of cyclin D1 gene expression (59-61), a reduction in its nuclear levels secondary to its sequestration in the cytoplasm by SLPI-enhanced expression of Ikappa Bbeta , might underlie the opposing consequence of SLPI on cyclin D1 promoter activity. Nevertheless, it is worthwhile to note that the changes in cyclin D1 transcriptional activity with SLPI are relatively modest. This finding coupled with the observation that the 8As subline with undetectable SLPI gene expression is competent to grow, although at a much diminished rate than cells with robust expression of this anti-protease, is indicative of a contributory rather than a critical role for SLPI in growth regulation. Consistent with this, mice null for SLPI are viable and do not exhibit gross phenotypic consequences (25).

Another potential (and novel) mechanism behind SLPI control of cell growth, as demonstrated here, may involve its effective inhibition of the expression of a number of growth suppressor and/or tumor suppressor genes. The latter include the following: 1) TGF-beta 1, a potent inhibitor of epithelial cell proliferation; 2) LOX, also known as ras-recision gene (51), which down-regulates the ras/mitogen-activated protein kinase signaling pathway (62, 63); and 3) IGFBP-3, a major factor in the control of IGF-I bio-availability and, hence, of its growth promoting activity and a pro-apoptotic agent that may be IGF-independent in this latter action (64). In this regard, we observed an inverse correlation between the expression of these genes and that of SLPI at the levels of mRNA (TGF-beta 1, LOX, IGFBP-3) or protein (IGFBP-3), although attempts to establish a causal relationship by the addition of rhSLPI to 8As cells in culture or by transient transfection of human SLPI expression construct to these cells did not yield the anticipated effects. Because these same treatments resulted in the induction of cyclin D1 gene expression, these findings suggest that the regulatory mechanisms underlying the control of the expression of these genes by SLPI, unlike that for cyclin D1, occur indirectly or via more long term changes in cell phenotype. For LOX, this is a likely possibility because it is a known transcriptional target of TGF-beta 1 (65). The intermediate steps leading to the inhibition of TGF-beta 1 and IGFBP-3 gene expression upon increased expression of SLPI cannot be deduced from the present studies, although a regulatory loop arising from TGF-beta 1 inhibition of SLPI gene expression, as demonstrated here, is consistent with reports observed for a bronchial epithelial cell line (66). In further agreement with the present results, a negative correlation between SLPI and TGF-beta 1 was reported in SLPI null mutant mice (25). In those studies, however, the consequence of the loss of SLPI expression was observed at the level of TGF-beta activation, rather than at the level of mRNA induction as reported here. Although the concentration of bioactive TGF-beta 1 was not measured in the present study, the parallel rise in protein (in vivo) and transcript (in vitro) levels of this growth inhibitor after ablation of the SLPI gene is clearly indicative of the physiological relevance of the SLPI/TGF-beta linkage.

Based on the observations from this study, we propose a model that illustrates the regulation by SLPI of cell proliferation via its positive and negative influence on the expression of genes with known growth regulatory activities (Fig. 9). In this model, SLPI activates cell proliferation directly through its control of cyclin D1 gene expression. SLPI inhibits the expression of the growth suppressors IGFBP-3 and TGF-beta 1, although the mechanism is likely indirect and may entail the participation of yet unknown intermediary steps. Furthermore, SLPI negatively regulates LOX (tumor suppressor) gene expression likely through a TGF-beta 1-mediated pathway. The end result of this multiple regulation is the synergistic induction of cell proliferation. Thus, despite the modest effect of SLPI on the expression of individual genes, the sum total of the collective SLPI actions is the generation of a significant growth stimulus.


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  		Fig. 9.   Model for regulation of endometrial epithelial cell proliferation by SLPI. The model illustrates that SLPI induces the expression of cyclin D1 and inhibits those of IGFBP-3, TGF-beta 1, and LOX. The designations are as follows: lines with arrow, positive regulation; blocked lines, negative regulation; solid lines, direct regulation; broken lines, indirect regulation; dotted lines, previously published data. The arrow around TGF-beta 1 indicates positive autoregulation.

Several important questions are raised by our results. One relates to the signaling pathway(s) by which SLPI promotes proliferation. Our finding that the addition of SLPI to culture medium results in the up-regulation of cyclin D1 gene expression is perhaps indicative of an integral membrane-localized SLPI receptor/binding protein. This may or may not be functionally related to a recently described SLPI-interacting protein identified by yeast two-hybrid screen as scramblase, an integral membrane protein of 35-37 kDa involved in movement of membrane phospholipids (67-69). However, in another report (70), SLPI bound to a monocyte membrane protein of 55 kDa. It has been noted that the three-dimensional structure of SLPI is suggestive of its ability to utilize membrane receptor(s) to mediate its biological actions (71); however, the definitive nature of this membrane component(s) is presently unknown. Furthermore, the similar inhibitory effects of TGF-beta 1 and SLPI on IGFBP-3 expression are not entirely consistent with their opposing consequences on cell proliferation, although this apparent discrepancy might be related to the nature of the parental cell line (Ishikawa) utilized in this study. In a number of adenocarcinoma cell lines, abnormal TGF-beta response could be generated in a gene-specific context (72-74). Finally, it would be of interest to ascertain whether other growth-regulatory genes mediate the proliferative effects of SLPI, and if their encoded products delineate sensitivity to SLPI in cell types other than the endometrial epithelial cells used here. Future studies in our laboratories utilizing the microarray and other approaches will address these questions.

In summary, the generation and subsequent use of an SLPI "knock-out" in vitro model has allowed for the initial elucidation of the molecular mechanism behind the proliferative function ascribed to SLPI. Our data suggest that this newly described pathway involves the up- and down-regulation by SLPI, respectively, of positive (cyclin D1) and negative (TGF-beta 1, LOX, IGFBP-3) growth-associated factors via direct and indirect mechanisms. Our results for TGF-beta 1 and IGFBP-3 are particularly intriguing because recent work (75) suggests a functional interaction of both systems to inhibit epithelial cell growth. In particular, IGFBP-3 appears to utilize the TGF-beta receptor/signaling systems (including Smads) to elicit its anti-proliferative effects. Our results place SLPI at the node of this interactive pathway as well as those for cyclin D1 (ras/MAPK and NF-kappa B) and LOX. Our findings may have physiological relevance to further understanding the growth control of normal epithelium as well as the lack thereof in certain epithelially derived carcinomas wherein high expression of SLPI is manifest.

    ACKNOWLEDGEMENTS

We thank the other members of our laboratories for helpful suggestions during the course of this work, and Drs. Chris Albanese and Richard G. Pestell (Albert Einstein College of Medicine, NY) and Dr. Peter S. Hiemstra (Leiden University, The Netherlands) for generous provision of reagents used in this study.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HD21961 and the Florida Agricultural Experiment Station (Publication Series No. R-08764).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Arkansas Children's Nutrition Center, Arkansas Children's Hospital Research Institute and Department of Physiology & Biophysics, University of Arkansas for Medical Sciences, Slot 512-20B, 1120 Marshall St., Little Rock, AR 72202. Tel.: 501-320-2859; Fax: 501-320-3161; E-mail: simmenfranka@uams.edu.

Published, JBC Papers in Press, May 22, 2002, DOI 10.1074/jbc.M203503200

    ABBREVIATIONS

The abbreviations used are: SLPI, secretory leukocyte protease inhibitor; BTEB1, basic transcription element-binding protein 1; LOX, lysyl oxidase; TGF-beta 1, transforming growth factor-beta 1; IGF, insulin-like growth factor; IGFBP-3, insulin-like growth factor-binding protein-3; MEM, minimal essential medium; FBS, fetal bovine serum; As, anti-sense; SSC, saline sodium citrate; MOPS, 3-N-morpholinopropanesulfonic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ddRT-PCR, differential display-reverse transcriptase-PCR; HBSS, Hanks' balanced salt solution; Me2SO, dimethyl sulfoxide; PBS, phosphate-buffered saline; PCNA, proliferative cell nuclear antigen; NRS, normal rabbit serum; NF-kappa B, nuclear factor-kappa B; TIMP, tissue inhibitor of metalloproteinase; rh, recombinant human.

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ABSTRACT
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RESULTS
DISCUSSION
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