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Endocrinology, doi:10.1210/en.2003-1261
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Endocrinology Vol. 145, No. 5 2245-2252
Copyright © 2004 by The Endocrine Society

Tumor Necrosis Factor-{alpha} Induces Serum Amyloid A3 in Mouse Granulosa Cells

Deok-Soo Son, Katherine F. Roby and Paul F. Terranova

Center for Reproductive Sciences (D.-S.S., K.F.R., P.F.T.) and Departments of Molecular and Integrative Physiology (D.-S.S., P.F.T.), Anatomy and Cell Biology (K.F.R.), and Obstetrics and Gynecology (P.F.T.), University of Kansas Medical Center, Kansas City, Kansas 66160

Address all correspondence and requests for reprints to: Dr. Paul F. Terranova, Center for Reproductive Sciences, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160-7417. E-mail: pterrano{at}kumc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TNF-{alpha} has significant inhibitory effects on steroidogenesis and folliculogenesis and is associated with several inflammatory responses. Because ovulation is an inflammatory reaction, the effects of TNF on the family of acute-phase proteins in granulosa cells were investigated. Granulosa cells from immature mice at 28 d of age were cultured in the presence of 10 ng TNF/ml for 24 h. Serum amyloid A3 (SAA3), a main acute-phase protein, was induced by TNF in granulosa cells. The other isoforms of serum amyloid proteins SAA1, SAA2, and SAA4 were neither expressed in granulosa cells nor induced by TNF. TNF did not induce SAA3 mRNA in granulosa cells from TNF receptor type 1 (TNFR1) knockout mice, although SAA3 mRNA was induced within 3 h after TNF treatment in wild-type cells. Two SAA3 promoters, –617/+73 and –198/+73, were responsive to TNF and to p65, a component of the TNF signaling molecule nuclear factor (NF)-{kappa}B. The –106/+73 promoter of SAA3 lacking a NF-{kappa}B-like site was not responsive to TNF or p65. In granulosa cells from TNFR1 knockout mice, the SAA3 promoter (–198/+73) was responsive to transfection with the p65 component of NF-{kappa}B, but neither TNF treatment nor overexpression of the p50 component of NF-{kappa}B increased promoter activity. Similar results were observed in the murine ovarian granulosa tumor cell line (OV3121-1). Overexpression of the inhibitor of NF-{kappa}B (called I{kappa}B) blocked SAA3 promoter activity induced by TNF and by p65 in OV3121-1 cells. Closer analysis of deletion mutants of the SAA3 promoter revealed the necessity of a NF-{kappa}B like site for responsiveness to TNF in the OV3121-1 cells. TNF rapidly increased p65 in OV3121-1 nuclei when compared with controls not treated with TNF. TNF also increased phospho-IkB and SAA3 in whole-cell homogenates as determined by Western blots. Thus, TNF likely increased SAA3 promoter activity and protein by activating NF-{kappa}B signaling via TNFR1 in mouse granulosa cells. SAA3 is a novel gene in granulosa cells with yet unknown functions in the ovary.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OVULATION HAS BEEN frequently compared with an inflammatory response (1, 2). Within a few hours of the LH surge, follicular hyperemia and edema occur and are mediated by vasoactive agents such as histamine, kinins, and prostaglandins. Cytokines are also involved in folliculogenesis, ovulation, and luteinization and modulate these processes (3). TNF-{alpha}, a multifunctional hormone-like polypeptide, modulates many genes involved in inflammation, infection, and malignancy. Macrophages are a main source of TNF in the ovary, and oocytes, corpora lutea, and theca and granulosa cells have been reported to contain TNF or its mRNA (4, 5, 6, 7). TNF is well known for its effects on steroidogenesis (8, 9, 10, 11), folliculogenesis (12, 13), ovulation (14), luteinization (15, 16), and fertility (17, 18) in numerous species including rodents and humans. TNF binding sites exist in granulosa cells (19, 20), and ovarian TNF signaling cascades regulating steroidogenesis act most likely via p55 TNF receptor type 1 (TNFR1) (18) rather than p75 TNFR2 (12, 18).

TNF, an IL-1-type cytokine, increases hepatic acute-phase proteins rapidly and abundantly, and the acute-phase proteins enhance protection against microorganisms and modify inflammation to reconstitute the homeostatic state (21). Although the liver is a well known source of acute-phase proteins, little is known concerning acute-phase proteins in the ovary. Serum amyloid A3 (SAA3) is expressed as an acute-phase protein in the rabbit ovary (22, 23); however, little is known regarding the site of synthesis and its regulation. Thus, the present study was designed to assess the effects of TNF on ovarian acute-phase proteins in mouse granulosa cells, focusing on signaling pathways involved in TNF-induced SAA3 using intact and TNFR1 knockout mice and a mouse granulosa tumor cell line.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Recombinant murine TNF (lyophilized from a 0.2-µm filtered solution in PBS containing 50 µg BSA/µg TNF) was obtained from R&D Systems (Minneapolis, MN). The following reagents were purchased from Sigma Chemical Co. (St. Louis, MO): penicillin G/streptomycin and fibronectin. Lipofectamine Plus, TRIzol, and all liquid culture media were acquired from Invitrogen (Grand Island, NY). Antisense and sense oligonucleotides of SAA isoforms were obtained from Integrated DNA Technologies (Coralville, IA). An inhibitor of nuclear factor (NF)-{kappa}B (I{kappa}B) expression vector and a nucleoporin antibody were obtained from BD Biosciences (Palo Alto, CA). Expression plasmids for the NF-{kappa}B components p65-pRc/RSV and p50-pcDNAI were kindly provided by Dr. Tom Maniatis (Harvard University, Cambridge, MA). A mouse ovarian granulosa tumor cell line (OV3121-1) was a gift from Dr. Kazuyoshi Yanagihara (National Cancer Center Research Institute, Tokyo, Japan). The SAA3 antibody was kindly given by Dr. Philipp Scherer (Albert Einstein College of Medicine, Bronx, NY). The p65 and ß-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The phospho-I{kappa}B (Ser 32/36) and I{kappa}B antibodies were purchased from Cell Signaling Technology (Beverly, MA). The luciferase reporter assay system was obtained from Promega (Madison, WI).

Animals
C57BL6 mice from Harlan, Inc. (Indianapolis, IN) and TNFR1 null mice on a C57BL6 background from Immunex (Seattle, WA) were established as breeding colonies in our laboratory. All mice were given commercial pellet feed and drinking water ad libitum and housed with controlled 12-h light, 12-h dark cycle under pathogen-free conditions. All handling of animals and procedures conformed to the guidelines set forth by the Institutional Animal Care and Use Committee of the University of Kansas Medical Center.

Cell cultures and treatments
Ovaries were collected under a laminar flow hood from mice at 28 d of age and placed in cold DMEM/F12 supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml). The ovaries were cleaned of all connective tissues and fat. Follicles were punctured using a 27-gauge needle attached to 1-ml syringe to extrude granulosa cells. Before culture, plates were coated overnight in 4 µg of fibronectin/ml Hanks’ solution. Then, granulosa cells (~2 x 105 cells/ml) were cultured at 37 C in a water-saturated atmosphere of 95% air and 5% CO2 in fibronectin-coated 12- or six-well culture plates with serum-free DMEM/F12 medium containing penicillin/streptomycin. After overnight culture to allow cellular attachment to the plate, the medium was removed and fresh medium was added. Treatments were initiated as outlined in Results.

OV3121-1 cells (~2 x 105 cells/ml) were also cultured under similar conditions as described above except for using RPMI 1640 medium (Mediatech Inc., Herndon, VA) containing 10% fetal bovine serum and penicillin/streptomycin to allow cellular attachment. For treatments, the RPMI 1640 medium was removed and serum-free DMEM/F12 medium containing penicillin/streptomycin was added. After overnight culture with serum-free media, treatments were initiated as outlined in Results.

DNA microarray
Total RNA was extracted using 1 ml TRIzol reagent per well (six-well plate) at room temperature for 5 min after which 200 µl chloroform was added, mixed, and centrifuged (12,000 x g for 15 min at 4 C). The aqueous phase was transferred to a fresh tube and 500 µl of isopropanol was added, and the tube was mixed, allowed to stand for 10 min, and centrifuged as before but for 10 min. The pellet was washed with 75% ethanol, centrifuged at 7000 x g for 5 min at 4 C, and the final pellet was saved as total RNA in RNase-free water. The cDNA template was reverse transcribed from total RNA using an oligo dT primer coupled with a T7 promoter and the Superscript Choice System (Invitrogen). In vitro transcription and biotin labeling of the cRNA target were produced from the cDNA and subjected to an Affymetrix mouse DNA microarray (Murine Genome U74Av2). Hybridization and washes were performed using the Affymetrix gene chip system as follows: hybridization for 16 h at 45 C and 60 rpm in the GeneChip Hybridization Oven 640 followed by automated washing and staining using the GeneChip Fluidics Station 400. Data analysis was conducted using the expression analysis software GeneSpring version 4.2, Data Mining Tool, MicroDB, or online with NetAffx Analysis Center at www.affymetrix.com. Absolute and comparison analyses were conducted using the following settings: scaling, all probe sets, target signal = 500; and normalization, user defined, scale factor = 1.

RT-PCR
Total RNA was isolated using TRIzol reagent as described above. The RT reaction conditions, using random primers with Maloney murine leukemia virus, were at 42 C for 60 min followed by 94 C for 10 min. Specific primers for SAA isoforms were designed as follows: 5'-GAA GGA AGC TAA CTG GAA AAA CTC-3' (sense) and 5'-CAG GCC CCC AGC ACA ACC TAC T-3' (antisense) for SAA1, 5'-ATG AAG GAA GCT GGC TGG AAA GAT G-3' (sense) and 5'-CTC AGG ACC CCA ACA CAG CCT TCT-3' (antisense) for SAA2, 5'-AGC CTT CCA TTG CCA TCA TTC TT-3' (sense) and 5'-AGT ATC TTT TAG GCA GGC CAG CA-3' (antisense) for SAA3, and 5'-GAG GTC TTG CTC GTG ATT CAC T-3' (sense) and 5'-TTT CTG GGT AGC CTG CAG GGT T-3' (antisense) for SAA4. L19 primers were used as a control (24). PCR was performed under the following conditions: denaturation at 94 C for 1 min, annealing at 58 C for 1 min, and extension at 74 C for 1 min with 25 cycles for SAA isoforms and 30 cycles for L19. Amplified PCR products were analyzed by electrophoresis in 2% agarose gels containing 1 µg ethidium bromide/ml. The fluorescent images were photographed under UV light.

Construction of the SAA3 promoter-luciferase gene
A 690-bp DNA fragment from –617 to +73 of the mouse SAA3 gene was generated by PCR using genomic DNA isolated from C57BL6 mouse liver. Primer sets were designed as follows: 5'-TAA CTC GAG GCC ACT TTC TGC CTG AA-3' for sense containing XhoI site and 5'-CCG AAG CTT GAA AGT TCT GGC AAC TC-3' for antisense containing HindIII site. The PCR was performed for 35 cycles at 94 C for 1 min, 55 C for 1 min, and 74 C for 1 min with a final extension at 74 C for 10 min. The amplified SAA3 DNA fragment was digested with XhoI and HindIII, and the fragment was loaded onto a spin-column containing a silica-gel membrane (Gel Extraction System, QIAGEN, Valencia, CA) that absorbed the DNA. The column was centrifuged at 12,000 x g for 1 min at room temperature followed by washing and elution with Tris buffer. The purified SAA3 promoter was subcloned into the XhoI and HindIII sites of the pGL3-basic vector. Deletion mutants were generated from the –617/+73 SAA3 promoter (mSAA3-617LUC) under the same PCR conditions using the following primers: 5'-GCA CTC GAG GAA GAC TTC AGA AAG TC-3' for –198/+73 deleted construct (mSAA3-198LUC) and 5'-GCG CTC GAG TAT CTT CTG AAA GAG AA-3 for –106/+73 deleted construct (mSAA3-106LUC). DNA sequences between –172 and –131 have core binding sites as follows (25): acute-phase response factor (APRF), Oct-1, NF-{kappa}B, two CCAAT binding proteins (C/EBP), nuclear factor for IL-6 expression (NFIL-6), and IL-6 response element-binding protein (IL-6REBP). Therefore, various deletion mutants to identify an important binding site regulating SAA3 were generated as follows (see Fig. 5Go): mSAA3-172LUC (all core binding sites), mSAA3-166LUC (a disrupted APRF site), mSAA3-163LUC (disrupted sites for APRF and Oct-1), mSAA3-159LUC (disrupted sites for APRF, Oct-1, and NF-{kappa}B), and mSAA3-143LUC (disrupted sites for APRF, Oct-1, NF-{kappa}B, NFIL-6, and one C/EBP). In addition, atypical NF-{kappa}B mutants of the SAA3 promoter of the same size (–172/+73) were generated as follows (see Fig. 6Go): GGAAATGCCT for atypical NF-{kappa}B site, GGAAAGTCCC for typical NF-{kappa}B site, and GCTAATGCCT for atypical mutant NF-{kappa}B site (underliningindicates different sequences from atypical NF-{kappa}B site).



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  		FIG. 5. Effect of TNF on the activity of SAA3 promoters containing core binding sites in OV3121-1 cells. Cells were transfected for 3 h with various mSAA3 promoters and then were incubated with or without TNF (10 ng/ml) for 24 h. The luciferase activity was normalized by total protein concentrations and expressed as a fold change by comparison with the control. Dark gray bars indicate significant increases (P <= 0.05) when compared with control. The data are from four experiments. The consensus sequences that differ from those between –172 and –131 are underlined. C, Control; T, TNF; LUC, luciferase. The characteristics of deletion mutants are as follows: mSAA3-172LUC with all core binding sites; mSAA3-166LUC with a disrupted APRF site; mSAA3-163LUC with disrupted APRF and Oct-1 sites; mSAA3-159LUC with disrupted sites for APRF, Oct-1, and NF-{kappa}B; and mSAA3-143LUC with disrupted sites for APRF, Oct-1, NF-{kappa}B, NFIL-6, and one C/EBP.

 


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  		FIG. 6. Effects of TNF on the activity of SAA3 promoters containing atypical, typical, and atypical mutant NF-{kappa}B binding sites in OV3121-1 cells. Cells were transfected for 3 h with various mSAA3 promoters and then were incubated with or without TNF (10 ng/ml) for 24 h. The luciferase activity was normalized by total protein concentrations and expressed as a fold change by comparison with the control. Dark gray bars indicate significant increases (P <= 0.05) when compared with control. The different consensus sequences are underlined. The data are from three experiments. C, Control; T, TNF; LUC, luciferase.

 
Transient transfection and the luciferase assay
Granulosa cells and OV3121-1 cells at approximately 50% confluency in 12-well plates were washed once with fresh DMEM/F12 (without additives) and were transiently transfected with vectors for 3 h at 37 C using 2 µl Lipofectamine and 5 µl Plus solution (Lipofectamine Plus, Invitrogen) per 500 µl media. Transfected cells were treated as outlined in Results and incubated for 24 h. After rinsing cells with ice-cold PBS and adding lysis buffer (Promega), cell lysates were centrifuged at 12,000 x g for 1 min at room temperature. The supernatant fluid was then used for determination of luciferase activity using a microplate luminometer. Luciferase activity expressed as relative light units was normalized to the protein level.

Western blot
Nucleic protein extracts and cell lysates were prepared, fractionated on SDS-polyacrylamide gels, and transferred to nitrocellulose membranes according to established procedures (26). Blocking of nonspecific proteins was performed by incubation of the membranes with 5% nonfat dry milk in Tris-buffered saline Tween 20 (TBST containing 10 mM Tris, 150 mM PBS, 0.05% Tween 20, pH 8.0) for 2 h at room temperature. Blots were incubated with primary antibodies at 1:1000 dilution in blocking solution overnight at 4 C. The membranes were washed three times with TBST for 10 min followed by incubation for 1 h with horseradish peroxidase-conjugated secondary antibody according to primary antibody used at 1:2500 in 5% milk/TBST. The membranes were then rinsed three times with TBST for 10 min, and the bands were visualized by enhanced chemiluminescence. After membrane stripping for 10 min with methanol containing 3% H2O2, nucleoporin and ß-actin were detected immunologically to serve as an internal loading control of nucleic protein extracts and cell lysates, respectively.

Statistics
Data were analyzed by Student’s t test and one-way ANOVA. If statistical significance (P <= 0.05) was determined by ANOVA, the data were further analyzed by the Student-Newman-Keuls method to detect specific differences between treatments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Type 1 and 2 acute-phase proteins in mouse granulosa cells
TNF increased SAA3 mRNA specifically and abundantly as determined by microarray (Table 1Go) and RT-PCR (Fig. 1Go). The mRNAs for SAA1, -2, and -4 were not detectable in mouse granulosa cells by either microarray or RT-PCR, and TNF was unable to increase the mRNAs as it did for SAA3 mRNA (Table 1Go and Fig. 1Go). By microarray, fibronectin was the only type 2 acute-phase protein detectable in granulosa cells, and TNF tended to decrease it (Table 1Go).


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  		TABLE 1. Effects of TNF on type 1 and 2 acute-phase proteins in mouse granulosa cells as determined by Affymetrix microarray

 


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  		FIG. 1. Effects of TNF (10 ng/ml) on SAA isoforms in mouse granulosa cells. Cells were incubated with vehicle (C) or TNF (T) for 24 h. After isolating total RNAs, RT-PCR was performed using specific primers for each SAA isoform. L19 was used as an internal housekeeping gene. M, Molecular marker in base pairs; +, C57BL6 mouse hepatic total RNA as a positive control for SAA isoforms. This experiment is representative of three experiments.

 
Time course for SAA3 and TNFR1 involvement
TNF increased SAA3 mRNA in cultured mouse granulosa cells to detectable levels within 3 h and the levels remained elevated throughout the 24 h culture period (Fig. 2AGo). However, granulosa cells from TNFR1 knockout mice did not respond to TNF in vitro after 24 h as evidenced by undetectable SAA3 mRNA (Fig. 2BGo).



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  		FIG. 2. A, The time course effects of TNF on SAA3 mRNA in cultured mouse granulosa cells from intact C57BL6 mice. Cells were incubated with TNF (10 ng/ml) for 0, 1, 3, 6, 12, and 24 h. B, Effects of TNF (10 ng/ml) on expression of SAA3 mRNA in granulosa cells cultured from intact (+/+) and TNFR1 knockout mice (–/–) for 24 h. Total RNA was isolated and RT-PCR was performed using primers for SAA3 and L19 as a loading control. M, Molecular marker in base pairs; C, control; T, TNF. This experiment is representative of two experiments.

 
TNF-induced SAA3 promoter activity in mouse granulosa cells and TNFR1 involvement
TNF increased luciferase activity of the mSAA3-617LUC and mSAA3-198LUC promoters in granulosa cells from intact mice, whereas TNF had no effect on activity of the mSAA3-106LUC promoter (Fig. 3AGo). These results indicated that TNF required a critical region between –198 and –106 in regulating SAA3 in mouse granulose cells. In contrast, TNF did not induce SAA3-198LUC promoter activity in granulosa cells of TNFR1 knockout mice (Fig. 3BGo).



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  		FIG. 3. A, Effects of TNF (10 ng/ml) on the luciferase activity of various SAA3 promoters in granulosa cells from intact C57BL6 mice; B, comparison of TNF response on the luciferase activity of SAA3 promoter (mSAA3-198LUC) in granulosa cells from intact (+/+) and TNFR1 knockout (–/–) mice. Cells were transfected for 3 h with SAA3 promoter luciferase reporter. Cells were incubated with or without TNF (10 ng/ml) for 24 h. The luciferase activity was normalized by total protein concentrations and expressed as a fold increase by comparison with the control. Dark gray bars indicate significant increase (P <= 0.05) when compared with control. This figure is the result of three experiments. C, Control; T, TNF; LUC, luciferase.

 
Dose-dependent effects of TNF on SAA3 mRNA and promoter activity in a mouse granulosa tumor cell line
In a mouse granulosa cell line (OV3121-1), TNF dose-dependently increased SAA3 mRNA (Fig. 4AGo) and promoter activity (Fig. 4BGo). The maximum induction of SAA mRNA was shown at 5 ng TNF/ml (Fig. 4AGo).



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  		FIG. 4. A, Dose-dependent effects of TNF on SAA3 mRNA in OV3121-1 cells (representative of two experiments). Cells were incubated with TNF (0, 0.1, 0.5, 1, 5, and 10 ng/ml) for 24 h. After isolating total RNA, RT-PCR was performed using primers for SAA3, and L19 was a loading control. B, Effects of TNF on SAA3 promoter activity in OV3121-1 cells (result of three experiments). Cells were transfected for 3 h with mSAA3-198LUC followed by incubation with or without TNF (10 ng/ml) for 24 h. The luciferase activity was normalized by total protein concentrations and expressed as a fold change by comparison with the control. Dark gray bars indicate significant increases (P <= 0.05) when compared with control (C).

 
Determination of the TNF-responsive site increasing SAA3 promoter activity in OV3121-1 cells: importance of an atypical NF-{kappa}B site
The mSAA3-172LUC containing all core binding sites, mSAA3-166LUC with a disrupted binding site for APRF, and mSAA3-163LUC with disrupted binding sites for APRF and Oct-1 were responsive to TNF (Fig. 5Go). However, the cells were not responsive to TNF when mSAA3-159LUC with disrupted binding sites for an APRF, Oct-1, and atypical NF-{kappa}B was tested (Fig. 5Go). Also mSAA3-143LUC with disrupted binding sites for APRF, Oct-1, atypical NF-{kappa}B, NFIL-6, and one C/EBP was not responsive to TNF (Fig. 5Go). The lack of response of the SAA3 promoter to TNF between –163 and –159 bp indicated the importance of an atypical NF-{kappa}B site in regulating promoter activity. TNF did not increase SAA3 promoter activity in OV3121-1 cells containing an atypical mutant NF-{kappa}B binding site, but TNF increased the activity of SAA3 promoters containing atypical and typical NF-{kappa}B binding sites (Fig. 6Go).

Effects of p65 and p50 expression vectors on SAA3 promoter activity in OV3121-1 cells: inhibitory effects of I{kappa}B and a rapid nuclear translocation of p65 by TNF
The NF-{kappa}B components, p65 and p50, were investigated as mediators of TNF signaling because it is well known that TNF increases NF-{kappa}B in several cell types (27). Overexpression of the p65 component of NF-{kappa}B also dose-dependently increased SAA3 promoter activity, whereas that of p50 had no effect on SAA3 promoter activity (Fig. 7AGo). The increase in SAA3 promoter activity induced by TNF and p65 was significantly reduced by cotransfection with an I{kappa}B expression vector, whereas I{kappa}B alone had no effect or a slight decreasing trend on basal promoter activity (Fig. 7BGo). Furthermore, TNF rapidly increased the nuclear localization of p65 within 10 min, and this was evident for up to at least 1 h (Fig. 7CGo).



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  		FIG. 7. A, Effects of p65 and p50 expression vectors on SAA3 promoter activity in OV3121-1 cells; B, effects of I{kappa}B on TNF- and p65-induced SAA3 promoter in OV3121-1 cells. Cells were transfected for 3 h with mSAA3-198LUC and cotransfected with either p65, p50, or I{kappa}B expression vectors. Where indicated, cells were incubated with or without TNF (10 ng/ml) for 24 h. The luciferase activity was normalized by total protein concentrations and expressed as a fold change by comparison with the control. C, Effects of TNF on p65 translocation in OV3121-1 cells. A and B represent data from three experiments each, whereas C was repeated three times and a representative figure is shown. Where indicated, cells were incubated with or without TNF (10 ng/ml) for 10 min, 30 min, and 1 h. Dark gray bars indicate significant increases (P <= 0.05) when compared with control (C). *, Significant decrease (P <= 0.05) when compared with p65.

 
Effects of p65 and p50 expression vectors on the luciferase activity of SAA3 promoters and effects of TNF on I{kappa}B and SAA3 protein in mouse granulosa cells: similarity with TNF response and phosphorylation of I{kappa}B
Overexpression of p65 increased luciferase activity of the mSAA3-617LUC and mSAA3-198LUC promoters in granulosa cells from intact mice, whereas it had no effect on activity of the mSAA3-106LUC promoter (Fig. 8AGo); this result was quite similar to TNF treatment (Fig. 3AGo). Overexpression of the p50 component of NF-kB did not increase promoter activity in OV3121-1 cells (Fig. 7AGo). Unlike TNF treatment, overexpression of p65 (but not p50) induced SAA3 promoter activity in granulosa cells of TNFR1 knockout mice (Fig. 8BGo). TNF increased phospho-I{kappa}B, a NF-{kappa}B regulating protein, and slightly decreased I{kappa}B protein and induced SAA3 protein in homogenates of granulosa cells from intact mice (Fig. 8CGo). On the other hand, this response to TNF was not evident in granulosa cells of TNFR1 knockout mice (Fig. 8CGo).



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  		FIG. 8. Effects of p65 and p50 expression vectors on the luciferase activity of SAA3 promoter in granulosa cells from intact (A, +/+) and TNFR1 knockout mice (B, –/–). Cells were transfected for 3 h with mSAA3-198LUC and cotransfected with either p65, p50, or I{kappa}B expression vectors. The luciferase activity was normalized by total protein concentrations and expressed as a fold change by comparison with the control. Dark gray bars indicate significant increase (P <= 0.05) when compared with control. C, Effects of TNF (10 ng/ml) on the expression of phosphorylated I{kappa}B (p-I{kappa}B), I{kappa}B, and SAA3 proteins in granulosa cells from intact (+/+) and TNFR1 knockout (–/–) mice. Where indicated, cells were incubated with or without TNF (10 ng/ml) for 24 h. Each gel lane was loaded with 20 µg of total protein using whole-cell lysates. ß-Actin was used as a loading control. A and B represent data from three experiments each, whereas C was repeated two times and a representative figure is shown. C, Control; T, TNF; LUC, luciferase.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The main findings in the present study were that TNF through TNFR1 induced SAA3 mRNA specifically and abundantly in mouse granulosa cells (Table 1Go and Fig. 1Go) and increased promoter activity involving a NF-{kappa}B-like site (Figs. 2Go, 3Go, and 5GoGoGoGo). Although mouse granulosa cells expressed a very low basal level of SAA3 mRNA (Table 1Go and Fig. 1Go), the other type 1 acute-phase protein mRNAs were not expressed and TNF did not alter these mRNAs (Table 1Go and Fig. 1Go). TNF appeared to decrease fibronectin mRNA, whereas the other type 2 acute-phase protein mRNAs were not detectable in mouse granulosa cells and not changed by TNF (Table 1Go). RT-PCR (Fig. 1Go) confirmed the specific induction of SAA3 mRNA by TNF among mRNAs of various SAA isoforms detected by microarray (Table 1Go). Several reports have shown that SAA3 is mainly expressed in extrahepatic organs including the whole ovary of rabbit (22, 23) or cell types such as macrophages and adipocytes (28, 29). In addition, using an SAA probe to recognize rabbit SAA1, SAA2, and SAA3 transcripts, SAA mRNA was expressed in unidentified cells in rabbit ovarian connective tissue after lipopolysaccharide (LPS) treatment and in atretic follicles (23). Interestingly, the SAA mRNA was not detectable after LPS in mink and mouse ovaries (23), indicating the possibility of interspecies differences in sensitivity to LPS.

The SAA3 mRNA was quickly induced by TNF within 3 h, and thereafter its steady-state level was maintained throughout 24 h of culture (Fig. 2AGo). SAA3 protein was evident at 24 h after TNF in vitro (Fig. 8Go). TNF did not alter SAA3 mRNA and protein in granulosa cells from TNFR1 knockout mice (Figs. 2BGo and 8CGo), indicating a requirement of TNFR1. TNF also increased the luciferase activity of mSAA3-617LUC and mSAA3-198LUC in granulosa cells from intact mice, whereas it had no effect on activity of mSAA3-106LUC (Fig. 3AGo). This indicates that at least the mSAA3-198LUC is a necessary and sufficient construct to induce SAA3 and that a critical regulatory region exists between –198 and –106. Several overlapping binding sites between –198 and –106 of the SAA3 promoter have been identified as follows: serum enhancer factor, atypical NF-{kappa}B binding, and C/EBP (25, 30, 31). These sites seemed to be necessary for the maximum induction of SAA3 mRNA, possibly by a cooperativity mechanism (25, 32, 33, 34).

The NF-{kappa}B components, p65 and p50, were investigated as effectors of SAA3 mRNA induction by TNF in mouse granulosa cells. Only p65 increased the luciferase activity of mSAA3-617LUC and mSAA3-198LUC but had no effect on that of mSAA3-106LUC (Fig. 8AGo). This is a similar pattern to that observed using TNF when an atypical NF-{kappa}B site was present with promoter activity or missing (no promoter activity) (Fig. 3AGo). The p50 vector was not active in increasing promoter activity in any experiments. A similar result using p50 was observed in hepatoma cell lines treated with IL-1 (32, 34). The failure of p50 to increase SAA3 promoter activity could be due to its lack of a functional transactivation domain, which is present in p65.

TNF did not increase the promoter activity of mSAA3-198LUC in granulosa cells from TNFR1 knockout mice, whereas p65 (but not p50) increased its activity (Fig. 8BGo). These data, coupled with the lack of responsiveness of SAA3 promoter when an atypical NF-{kappa}B binding site was missing, indicate that TNF-induced SAA3 requires p65 to be activated via TNFR1. However, the increase in SAA3 promoter activity induced by TNF and p65 may use additional mechanisms, although the NF-kB site appears to be common to both.

The mouse granulosa tumor cell line (OV3121-1) supported the results observed in the primary culture of mouse granulosa cells. TNF induced dose dependently SAA3 mRNA in OV3121-1 cells (Fig. 4AGo). In addition, TNF and the p65 expression vector, each alone, increased luciferase activity of mSAA3-198LUC in a dose-dependent manner, but the p50 vector was ineffective (Figs. 4BGo and 7AGo). Cotransfection with the I{kappa}B vector decreased the TNF- and p65-induced SAA3 promoter activities in OV3121-1 cells (Fig. 7BGo). In addition, disruption of the atypical NF-{kappa}B site prevented TNF responsiveness of the SAA3 promoter (Figs. 5Go and 6Go). These observations strengthen the link for involvement of the p65 component of NF-{kappa}B in TNF induction of SAA3 mRNA in mouse granulosa cells because I{kappa}B inactivates NF-{kappa}B and the activity of SAA3 promoter requires an atypical NF-{kappa}B site. It is well recognized that TNF activates NF-{kappa}B (27) and also that TNF activated NF-{kappa}B in rat granulosa cells (35). TNF caused a rapid (10 min) appearance of p65 component of NF-kB in the nucleus of OV3121-1 cells (Fig. 7CGo). In the granulosa cell, it appears that TNF increased phospho-I{kappa}B, which caused release of NF-kB from the Ik-B/NF-kB complex. The released NF-kB then binds to an atypical NF-kB site on the promoter of SAA3 causing activation of the promoter.

The physiological roles of TNF-induced SAA3 in ovarian granulosa cells are unknown. Because SAA3 is a high-density apolipoprotein (28), it may modulate cholesterol transport and metabolism in microenvironments within the ovary as observed by SAA1 and SAA2 in hepatic cells (36, 37). Furthermore, SAA3 has been shown to induce extracellular matrix-degrading enzymes such as collagenase in rabbit synovial fibroblasts (38, 39) and matrix metalloproteinases in rabbit chondrocytes (40). These two enzymes are thought to be involved in ovarian functions including follicular development and rupture (41, 42). In addition, human SAA inhibited IL-1- and TNF-induced fever in mice (43), suggesting that SAA3 may also be a modulator of ovarian inflammatory events such as ovulation. Female TNFR1 knockout mice have several reproductive alterations including early puberty as determined by vaginal opening, ovulation of more ova in response to exogenous gonadotropins, and early termination of ovarian cycles (18). Although some of these may be related to the lack of TNF stimulation of SAA3, other cytokines such as IL-1 may replace this function of TNF. Additional investigations are required to determine the exact roles of SAA3 in ovarian function.

In summary, TNF increased SAA3 promoter activity, mRNA, and protein in mouse granulosa cells, and this was mediated by TNFR1. In addition, TNFR1-dependent activation of the p65 component of NF-{kappa}B increased SAA3 promoter activity in mouse granulosa cells, possibly through interaction with an atypical NF-{kappa}B binding site.


   Acknowledgments
 
We thank Dr. Tom Maniatis (Harvard University, Cambridge, MA) for p65-pRc/RSV and p50-pcDNAI vectors, Dr. Kazuyoshi Yanagihara (National Cancer Center Research Institute, Tokyo, Japan) for OV3121-1 cells, and Dr. Philipp Scherer (Albert Einstein College of Medicine, Bronx, NY, for SAA3 antibody).


   Footnotes
 
This research was supported by grants from the National Cancer Institute (CA50616 to P.F.T.), National Institute of Child Health and Human Development (Specialized Cooperative Center Program in Reproduction Research, U54HD33994 to P.F.T.), and a Kansas Biomedical Research Infrastructure Network Grant from the National Center for Research Resources, National Institutes of Health (RR 16475).

Abbreviations: APRF, Acute-phase response factor; C/EBP, CCAAT binding proteins; I{kappa}B, inhibitor of NF-{kappa}B; IL-6REBP, IL-6 response element-binding protein; LPS, lipopolysaccharide; NFIL-6, nuclear factor for IL-6 expression; NF-{kappa}B, nuclear factor-{kappa}B; SAA3, serum amyloid A3; TBST, Tris-buffered saline with Tween 20; TNFR1, TNF receptor type 1.

Received September 19, 2003.

Accepted for publication January 21, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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