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Regular Article |
-Initiated Signaling in Bovine Luteal Cells1
a The Women's Research Institute, Wichita, Kansas 67214-3199
b Department of Obstetrics and Gynecology, University of Kansas School of Medicine, Wichita, Kansas 67214-3199
c Veterans Affairs Medical Center, Wichita, Kansas 67218
d Vincent Center for Reproductive Biology, Massachusetts General Hospital, Boston, Massachusetts 02114
ABSTRACT
Interferon gamma (IFN
) has been implicated as a mediator of luteal steroidogenesis and cell fate. IFN-initiated signaling events, although implied by studies in cell lines, have yet to be described in primary luteal cells. The objective of these studies was to begin to characterize IFN-initiated signaling within luteal cells. Dispersed bovine luteal cell cultures were challenged with increasing levels of bovine recombinant IFN (0–1000 U) or IFN (200 U) in the presence or absence of tumor necrosis factor 
(TNF, 10 ng/ml) over time (short term, 0–60 min; long term, 0, 24, 48 h). Fractionated or total cell lysates were evaluated by the Western blotting technique to determine the changes in the levels of signal transducers and activators of transcription (STAT), interferon regulatory factor 1 (IRF-1), and I kappa B (I
B-). Utilizing antibodies that recognize the nonphosphorylated forms of STAT-1 and STAT-3, it was determined that levels of STAT-1 and STAT-3 in total cell lysates were constitutively expressed and did not change in response to treatment with IFN or TNF. In contrast, nuclear levels of STAT-1 and phosphorylated STAT-3 were elevated in a time-dependent manner in response to IFN treatment. Furthermore, IFN and TNF treatment elevated levels of IRF-1 within 2 h. TNF-induced increases in the levels of IRF-1 were transient, whereas the levels of IRF-1 in response to IFN treatment remained elevated at 48 h. These data suggest that IFN treatment can activate members of the STAT pathway, resulting in increased levels of IRF-1. TNF treatment induced a rapid decrease in the [bu791]levels of IB-. IFN treatment did not alter the levels of IB- and failed to inhibit the TNF-initiated decrease in the levels of IB-. The present experiment demonstrates that the steroidogenic cells of the corpus luteum have the capacity to respond to IFN via activation of STAT and IRF-1, providing further evidence that IFN may be involved in the luteolytic process. These data also suggest that IFN does not signal through the nuclear factor B cell survival signaling pathway.
corpus luteum, corpus luteum function, cytokines, signal transduction
INTRODUCTION
Interferon gamma (IFN), a cytokine derived from T lymphocytes [1], has been implicated in the luteolytic process [2–6]. Evidence for the luteolytic actions of IFN is supported by a number of in vitro studies wherein treatment of luteal cells with IFN resulted in elevated levels of prostaglandin F2 [2], inhibition of progesterone [2–6], increased levels of class II major histocompatibility complex (MHC) antigens [7], and cell death [3]. Moreover, the cytotoxic effects of IFN were enhanced in the presence of tumor necrosis (TNF) [3]. Although a synergistic relationship between IFN and TNF is implied, the complexities of their individual signaling pathways and their interactions in the corpus luteum (CL) are unknown.
IFN signals through the janus activated kinases (JAK) and signal transducers and activators of transcription (STAT). Receptor-ligand binding activates JAK proteins, which phosphorylate tyrosine residues on one or more of the STAT proteins [8]. Phosphorylation of STAT-1 results in the formation of STAT-1 homodimers (also known as gamma interferon activation factor, GAF) [9], which translocate to the nucleus where they bind specific DNA sequences known as gamma-activated sites (GAS) [10]. Binding of STAT-1 dimers to the GAS elements can mediate the transcription of IFN-responsive genes, including interferon regulatory factor 1 (IRF-1), a secondary transcription factor readily inducible by IFN [11]. There are also some reports suggesting that TNF can induce IRF-1 in specific cell types [12]. IRF-1 can mediate the transcription of genes bearing IFN-stimulated response elements (ISREs) within their promoters. Furthermore, most genes synergistically inducible by TNF and IFN contain ISRE and nuclear factor B (NFB) sites in their promoter regions [13]. IFN and TNF also have the capacity to signal through the NFB signaling pathway [14], which is considered a prosurvival pathway. Therefore, it is feasible that IFN and TNF can activate multiple signaling pathways. This notion is supported by studies in a number of cell lines, yet there is little known with respect to IFN-initiated signaling events in the primary steroidogenic cells of the CL. The following study was designed to characterize the mediators of IFN-initiated signaling events in the steroidogenic cells of the CL.
MATERIALS AND METHODS
Animal Model
Corpora lutea from cows in early pregnancy (2–3 mo gestation based on fetal crown-rump length; of >9 to <30 cm) were obtained from a local slaughter facility in accordance with protocols approved by the local institutional animal care and use committee. Corpora lutea were dispersed with collagenase as previously described [15]. Prior to plating, 60-mm tissue culture dishes (Falcon; Fisher, St. Louis, MO) were pretreated with 10% fetal calf serum in medium 199 (M199; supplemented with 0.1% BSA, 25 mM Hepes, 25 mM NaHCO3, 100 U/ml penicillin G, 100 µg streptomycin, and 25 µg/ml gentamicin) for 1 h. The medium was aspirated, and the dishes were rinsed with M199. Luteal cells (105/cm2) were incubated in serum-free M199 (supplemented with 0.1% BSA, 25 mM Hepes, and 25 mM NaHCO3) containing 5.0 µg/ml insulin, 5.0 µg/ml transferrin, and 0.005 µg/ml selenium (ITS; Beckton-Dickinson, Bedford, MA) in an atmosphere of humidified air with 5% CO2 at 37°C. Cells were allowed to attach to the culture dishes for at least 24 h. After the initial incubation period, the cells were washed and the medium was replaced with fresh serum-free medium (supplemented with 0.1% BSA and ITS). Using this method, luteal cell cultures contain predominantly small and large luteal cells and few if any endothelial cells. The data presented are from a minimum of three separate luteal cell preparations, each obtained from a single CL derived from a different animal unless otherwise stated.
Treatments
Prior to treatment with IFN or TNF, the medium was aspirated and replaced with fresh medium and the cells were allowed to equilibrate for a minimum of 3 h. The initial experiment involved treatment of luteal cell cultures with increasing concentrations of IFN (0, 10, 100, 200, 500, 1000 U) for 15 or 20 min to establish the optimal concentrations of IFN to be used for subsequent experiments. Based on the results from this experiment, all following experiments utilized IFN at a concentration of 200 U/ml. Based on our previous studies [16], we used TNF at a concentration of 10 ng/ml. IFN treatment effects were determined in short-term (0, 2, 5, 10, 15, 30, and 60 min) and long-term (0, 1, 4, 8, 12, and 24 h) cell cultures. In addition, the effects of IFN (200 U/ml) alone or in combination with TNF (10 ng/ml) were analyzed in long-term culture (48 h). For all experiments, nontreated cell preparations were analyzed as internal controls.
Preparation of Nuclear and Total Cellular Lysates
Incubations were terminated by aspiration of the medium followed by the addition of lysis buffer (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Trition X-100, 0.5% NP-40) for total cellular lysates and TSE buffer (10 mM Tris, pH 7.4, 0.25 M sucrose, 0.1 mM EDTA) for nuclear lysates. Both buffers contained the following proteinase and phosphatase inhibitors: 50 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 µg/ml pepstatin A. Cells were scraped from culture dishes and transferred to bullet tubes. Nuclear lysates were homogenized with a pellet pestle motor (Kimble/Kontes, Vineland, NJ) and subsequently centrifuged at 1000 x g for 15 min at 40°C. The supernatant was discarded, and the nuclear pellet was resuspended in lysis buffer. All lysates were stored at -20°C until assayed.
Western Blotting Procedures
Total cellular lysates and nuclear lysates (10–20 µg/lane) were subjected to electrophoresis on SDS-polyacrylamide gels under reducing conditions utilizing a 5% stacking gel (pH 6.8) and either a 10% or a 12% resolving gel (pH 8.8) at 15 mA for 3 h at room temperature. Resolved proteins were subsequently transferred at 75 V for 2 h at 4°C to an Immobilon-P polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA) in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol). The membranes were stained with Ponceau for confirmation of equal protein loading. Immunoblotting was performed using commercially available antibodies: rabbit anti-STAT-1, rabbit anti-IRF-1, and rabbit anti-IB- (Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-STAT-3 and rabbit anti-pSTAT-3 (New England Biolabs, Beverly, MA). Membranes were blocked for 1 h in PBST (100 mM NaPO4, 100 mM NaCl, 0.05% Tween-20, pH 7.4) containing 5% nonfat dry milk for the detection of STAT-1, STAT-3/pSTAT 3, and IRF-1. Western blotting buffer (WBB: 0.15 M NaCl, 1 mM EDTA, 0.05 M Tris-HCl, pH 7.4, 0.05% NP-40, 0.1% BSA) was substituted for PBST in the blocking of IB-. Primary antibodies were administered in their respective blocking buffers as follows: anti-STAT-1 (11000) for 2 h, anti-STAT-3 and pSTAT-3 (1:1000) for 1 h, anti-IRF-1 (1:1000) overnight, and anti-IB- (1:500) overnight. Secondary antibody anti-rabbit IgG/horseradish peroxidase was added for 1 h in the respective blocking buffers at following dilutions: 1:5000 (anti-STAT-1 and IRF-1) and 1:2000 (anti-STAT-3, pSTAT-3, and IB-). All membranes were subjected to three 10-min washes in either PBST or WBB following both primary and secondary antibody incubations. The blots were visualized using enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, Piscataway, NJ). Multiple exposure times (5 sec to 5 min or longer as required) were used to maximize visualization. Bands that were visible on the autoradiographs were quantitated using scanning densitometry with Kodak 1-D image analysis software.
Cell Morphology
To determine whether treatment with the cytokine(s) had an effect on cell morphology, the number of apoptotic cells was counted in the control, TNF, IFN, or TNF + IFN cultures at 24 and 48 h as previously described [16]. The medium was aspirated, and the cells were fixed with 4% paraformaldehyde and stained with a DNA-specific fluorochrome (Hoechst-33258; Sigma Chemical Co., St. Louis, MO). The number of apoptotic cells was determined by calculating the mean number of apoptotic nuclei in a total of 10 separate fields within each well on a chamber slide. The percentage of apoptotic cells in control or cytokine-treated cultures was calculated by dividing the mean number of apoptotic cells by the mean total number of cells.
Determination of Progesterone Concentration in the Cultured Medium
The medium was collected at the termination of all experiments and stored at -20°C for steroid analysis. The concentration of progesterone in the medium was determined by RIA utilizing standardized methods previously validated and described [16].
Data Analysis and Quantitation
Quantitative data (combined results from the replicate experiments) were analyzed as raw data, log-transformed data, or percentage of maximum by one-way analysis of variance followed by the Duncan new multiple range test or the Student t-tests for paired comparison. The results were expressed as the mean ± SEM. A value of P < 0.05 was considered significant.
RESULTS
IFN Dose Response Effect on the Levels of STAT-1 and pSTAT-3
Treatment of cultured bovine luteal cells with increasing concentrations of IFN for 20 min resulted in dose-dependent increases in the nuclear levels of STAT-1 and phosphorylated STAT-3 (pSTAT-3, Fig. 1, n = 2). Based on the results from this experiment, the 200 U/ml concentration was used for subsequent experiments. The final concentration of recombinant IFN utilized in this study was similar to that previously used by others in follicular granulosa cell studies [17].
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Effect of IFN or TNF Treatment on the Levels of STAT-1 and pSTAT-3
Western blot analysis indicated that IFN treatment (0–60 min) did not alter the levels of nonphosphorylated STAT-1 in total cell lysates (Fig. 2; n = 5). However, analysis of nuclear lysates following treatment with IFN indicated that levels of STAT-1 were elevated (P < 0.05, n = 4) 5.7-, 5.0-, and 4.6-fold at 15, 30, and 60 min, respectively, compared with the 0-min controls (Fig. 3). Levels of STAT-1 in the nuclear lysates from 0-min control samples were not different from those in the 60-min control samples. Similar to the situation for STAT-1, no change was observed in the levels of nonphosphorylated STAT-3 in total cell lysates over time following treatment with IFN (Fig. 2). Treatment with IFN did, however, increase the levels of phosphorylated STAT-3 (pSTAT-3) within the nuclear fractions at 15 and 60 min (106- and 131-fold increase, respectively; n = 3, P < 0.01) over the levels in the nontreated 0-min samples (Fig. 4). Levels of pSTAT-3 in the nuclear lysates derived from the 0-min control samples were not different from the levels in the 60-min control samples. Treatment of luteal cell cultures with TNF had no effect on the nuclear levels of STAT-1 or pSTAT-3 (data not shown).
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Effect of IFN or TNF Treatment on the Levels of IRF-1
The levels of IRF-1 were elevated over controls in response to IFN. A significant induction (approximately 10-fold) was evident at 8 h (P < 0.05) (Fig. 5; n = 4), which remained elevated at 48 h (Fig. 6; n = 4). Treatment with TNF (10 ng/ml) also increased IRF-1 approximately sixfold by 2 h (P < 0.05) as compared with controls (Fig. 7; n = 5). TNF-induced IRF-1 levels remained elevated at 8 h (P < 0.05) but were undetectable at 24 and 48 h (data not shown).
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Effect of IFN and TNF Treatment on the Levels of IB-
Treatment with TNF initiated a time-dependent decrease in the levels of IB- (Fig. 8A), whereas IFN had no effect on the levels of IB- protein over time (0–20 min) (Fig. 8B). No difference was observed between the samples at Time 0 and those collected 2 and 5 min after treatment; however, within 10 min of TNF treatment, IB levels declined (64% ± 16.5%, n = 3, P < 0.01) when compared with the 0-min control (Fig. 8). The levels of IB- decreased (82% ± 3%, n = 3, P < 0.01) by 20 min when compared with the 0-min control. The combined treatment with TNF and IFN yielded results similar to those observed following treatment with TNF alone (Fig. 8C). A significant reduction in the levels of IB- (50% ± 13.0%, n = 3, P < 0.01) was observed by 5 min after treatment. The levels of IB- were further reduced (P < 0.001, n = 3) at 10 min (89% ± 7.8%) and 20 min (96% ± 4.5%) when compared with the 0-min control values.
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Effect of IFN and TNF Treatment on Luteal Cell Morphology
A significant increase (P < 0.05, n = 3) in the number of apoptotic cells was observed at 48 h in cultures treated with IFN (14.5% ± 1.1%) or with the combination of IFN and TNF (18% ± 1%) when compared with the number of apoptotic cells observed in the control (10% ± 0.3%) or the TNF-treated (10% ± 0.0%) cultures.
Effect of IFN and TNF Treatment on Progesterone Secretion
A significant decrease (P < 0.05, n = 3) in the level of progesterone at 48 h was observed in cultures treated with IFN (147 ± 22 ng/ml, n = 7) or with the combination of IFN and TNF (98 ± 15 ng/ml, n = 8) when compared with the levels of progesterone detected in the medium derived from the control (192 ± 23 ng/ml, n = 10) or TNF-treated (186 ± 25 ng/ml, n = 10) cultures.
DISCUSSION
IFN-regulated gene transcription is mediated by the phosphorylation and nuclear translocation of STAT-1 [8, 13, 18, 19]. STAT-1-homodimers (GAF) are known to initiate the transcription of IFN-responsive target genes, including IRF-1. The STAT-1 homodimer translocates to the nucleus where it binds to DNA sequences in the promoter region of target genes, initiating gene transcription [13]. In the present study, IFN activated STAT-1 and IRF-1 in primary luteal cells. These data extend previous observations that suggested that the steroidogenic cells of the CL are responsive to IFN [2–6].
Although IFN primarily activates STAT-1 [8, 13, 18, 19], nuclear levels of pSTAT-3 were also elevated in primary luteal cell cultures. Activation of STAT-3 by IFN has also been reported in non-ovarian cell types, including 3T3-L1 rat adipocytes [20, 21]. The role of IFN-activated STAT-3 in luteal cells is unknown. Based upon the data obtained in other model systems, STAT-3 is involved in interleukin-6-mediated suppression of apoptosis in T cells of transgenic mice [22, 23] and is important in development; STAT-3-deficient mice die early in embryogenesis [24]. Recently, it has been shown that STAT-1 and STAT3 have opposing actions on the Bcl-2 and Bcl-x promoters. For example, IFN stimulation of U3A-ST1 cells, which overexpress STAT-1, results in reduced expression of Bcl2, whereas CT-1, a STAT-3 activator, can enhance Bcl-2 and Bcl-X activity [25]. Although altered expression of Bcl-2 family members has been implicated in luteolysis [26], whether or not the IFN can alter the transcription of Bcl2 family members in luteal cells has yet to be determined. From the studies performed in nonovarian tissues and cells, it is evident that activated STATs can mediate a number of cellular responses. Moreover, recent studies utilizing cultured immature rat granulosa cells, cultured differentiating rat granulosa/luteal cells, or the pseudopregnant rat model have demonstrated that treatment with prolactin, a luteotrophic hormone, can result in increased phosphorylation of STAT-3 and/or STAT-5 [27–29]. These studies provide evidence that STAT-3 and STAT-5 may have a significant role in luteal formation. However, the physiological relevance of STAT activation within the CL during luteolysis remains to be determined.
IRF-1 is a primary response gene induced by IFN via the STAT-mediated activation of GAS located within the IRF-1 promoter [10]. In the present study, the levels of IRF1 protein were elevated by treatment with IFN and were maintained throughout the time points evaluated. IRF-1 can bind to ISREs and regulate transcription of additional IFN-responsive genes [13]. IRF-1 also can mediate the levels of MHC antigens [13, 20]. In one study [7], IFN treatment resulted in an increase in MHC class I antigens in bovine luteal cells. Furthermore, IFN treatment is known to upregulate levels of TNF receptor 1 mRNA [30, 31], presumably mediated via increases in IRF-1. Consistent with results derived from previous studies [12, 32], treatment of luteal cell cultures with TNF or IFN alone resulted in elevated levels of IRF-1. Although TNF treatment increased levels of IRF-1, the TNF-stimulated levels were no longer evident after 24 h. In contrast, IRF-1 levels remained elevated following 24 or 48 h of stimulation with IFN. Treatment with TNF and IFN also resulted in elevated levels of IRF-1 similar to those produced by IFN treatment alone. The differential, functional, and cell death responses observed previously [2–6, 17] as a result of TNF and IFN treatment, either individually or as a combined treatment, may be explained in part by the transient increase in IRF-1 observed in response to TNF as opposed to the sustained induction of IRF-1 following stimulation with IFN.
The active form of STAT-1 reportedly exists for up to 2 h before it is thought to be ubiquitinated and targeted for proteolysis [33, 34], and yet IRF-1 levels were maintained at an elevated state at 48 h, presumably in response to activated STAT-1. IRF-1 levels may be maintained by a positive feedback loop, or STAT-1 induction of IRF-1 may be accompanied by the activation of additional genes or inhibitors that potentially impede the degradation of IRF-1. The transient levels of IRF-1 following TNF stimulation may, however, be the result of a negative feedback loop or the activation of proteolysis. Whether the TNF transient induction of IRF-1 occurs in the presence of IFN and whether IFN can alter TNF induction of IRF-1 remains to be determined.
The synergistic death effect of TNF and IFN in luteal cells [3] may be mediated by other mechanisms. For example, activation of the transcription factor NFB may be in part mediated by the combined actions of TNF and IFN. TNF interaction with its receptor can initiate the formation of a receptor-cytoplasmic complex [35–37]. Formation of the TNF receptor-associated death domain, TNF receptor-associated factor, receptor-interacting protein, and NFB-inducing kinase complex leads to the activation of the IB kinase complex, which subsequently phosphorylates IB- [14]. Phosphorylation of IB- presumably results in its ubiquitination and subsequent degradation by the 26S proteasome, thereby releasing NFB to translocate to the nucleus to mediate transcription of genes containing NFB sites in their promoter regions, including antiapoptotic genes [38, 39].
TNF treatment reduced levels of IB- in luteal cells as early as 10 min after treatment. In contrast, IB- levels remained constant in response to IFN treatment; however, costimulation with TNF and IFN resulted in decreased levels of IB- similar to those observed in response to TNF treatment alone. The inability of IFN alone to decrease the levels of IB- alone suggests that IFN does not signal directly through the NFB signaling pathway within cultured bovine luteal cells. These results contradict those of previous studies, in which IFN treatment resulted in the activation of NFB [14]. The findings in the present study, however, do not negate the potential involvement of NFB with regard to alternative cell signaling mechanisms as a result of the synergistic actions of TNF and IFN. The combined effect of TNF and IFN treatment frequently may contain both NFB binding sites and ISREs [13]. Thus, the effects observed in luteal cells in response to IFN and TNF may be the sum of multiple signaling pathways and therefore multiple protein-DNA interactions. Additional studies will be necessary to further verify the activation and subsequent interaction of NFB and STAT dimers with their respective binding sites as a function of TNF and IFN stimulation in luteal cells.
ACKNOWLEDGMENTS
The authors thank Dr. Dale Godson (Veterinary Disease Organization, Saskatchewan, Canada) for providing the recombinant bovine INF for this project.
FOOTNOTES
First decision: 18 October 2000.
1 Supported in part by NIH grant R01-HD35934 to B.R.R. and J.S.D. 
2 Correspondence: Bo R. Rueda, Vincent Center for Reproductive Biology, Massachusetts General Hospital, VBK137E-GYN, 55 Fruit St., Boston, MA 02114. FAX: 617 726 7548; brueda{at}partners.org
Accepted: December 27, 2000.
Received: September 6, 2000.
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