Tissue-Protective Effects of Estrogen Involve Regulation of Caspase Gene Expression

David G. Monroe1, Ryan R. Berger and Michel M. Sanders

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455

Address all correspondence and requests for reprints to: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 6-155 Jackson Hall, 321 Church Street Southeast, Minneapolis, Minnesota 55455. E-mail: sande001{at}umn.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen plays a critical role in the protection from apoptosis in several cell types because the withdrawal of estrogen leads to increased apoptosis in tissues such as the brain, endothelium, testes, and uterus. Our recent report demonstrated that the chick oviduct also regresses through apoptotic mechanisms during estrogen deficiency. Despite these observations, very little is known concerning the intracellular mechanisms by which estrogen opposes apoptosis. To better understand how estrogen exerts its antiapoptotic effects, several key apoptotic genes were examined for their regulation by estrogen. Our results show that mRNA expression levels of Bcl-2, hsp-70, c-myc, Bcl-Xl, caspase-3, and caspase-6 remain essentially constant when apoptosis is stimulated by estrogen withdrawal. However, the genes for caspase-1 and caspase-2 are rapidly stimulated, at least for the most part, at the transcriptional level after the withdrawal of estrogen. This increase in caspase-2 mRNA is followed by an increase in enzyme activity. Furthermore, although mRNA expression levels are unaffected, both caspase-3 and caspase-6 proenzymes are activated in the estrogen-withdrawn cells. Taken together, these results demonstrate that estrogen has the potential to oppose apoptosis by regulating caspase activity through both transcriptional and posttranscriptional mechanisms in reproductive tissues.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
APOPTOSIS IS A stereotyped form of cell death triggered by a variety of intracellular and extracellular signals such as Fas-ligand binding, UV irradiation, overexpression of proapoptotic genes, and growth factor withdrawal. Some of the characteristic features of apoptosis are DNA condensation and fragmentation, changes in membrane composition, and cytoplasmic shrinkage (1). The molecular events involved in apoptosis are largely mediated by specific cysteine proteases called caspases (reviewed in Ref. 2). Caspases share significant homology with the Caenorhabditis elegans ced-3 protein, which is involved in apoptosis during development (3). Before the induction of apoptosis, caspases are widely expressed and exist as inactive proenzymes (or procaspases). The inactive procaspases are proteolytically activated by cleavage at specific amino acid sequences to form active enzymes. Generally, the apoptotic process is initiated by the activation of one or more initiator caspases (caspases-1, -2, -8, -9, and -10) (reviewed in Ref. 4). The specific initiator caspase used can be dependent on the apoptotic stimulus and the type of apoptotic pathway present in the cell. Regardless of which pathway is active, the initiator caspases activate one or more executioner or effector caspases (caspases-3, -6, and -7) by cleavage of their proenzymes. The effector caspases bring about the successive dismantling of the cell by cleaving intracellular substrates such as poly-(ADP-ribose) polymerase, specific cytoskeletal proteins, and nuclear lamins (5, 6).

Steroid hormones have long been known to modulate apoptosis in many different cell types (7). Early studies linking steroid hormones to apoptosis demonstrated that glucocorticoids cause apoptosis in thymocytes and that this process is dependent on glucocorticoid receptor concentrations (8). On the other hand, the sex steroids generally have been shown to demonstrate protective effects against apoptosis. For example, removal of androgens from the prostrate or progesterone from the uterine epithelial cells results in apoptosis of those cell types (8). In addition, estrogen plays a critical role in protection from apoptosis in neuronal, endothelial, and testicular cells because these cell types undergo apoptosis when deprived of estrogen (9, 10, 11, 12). The protective effects of estrogen are also evident in vivo during normal reproductive cycling because the uterus undergoes apoptosis when estrogen levels are low (12, 13). This suggests that estrogen plays a critical role in uterine homeostasis by preventing apoptosis when it is present, and conversely, allowing apoptosis when it is withdrawn.

The activation of the caspase cascade is one of the commitment steps leading to apoptosis. Therefore, the ability of a cell to control caspase activation in response to environmental cues is critical to its survival or death. How estrogen prevents caspase activation, and therefore apoptosis, is unclear. Numerous genes may be involved in the decision of a cell to undergo apoptosis, providing several potential targets for estrogen to affect the cell death pathway. For example, the antiapoptotic gene Bcl-XL is stimulated by estrogen in neuronal cells, leading to cell survival (14). This suggests that the withdrawal of estrogen, as in menopause, would result in decreased Bcl-XL levels and increased neuronal apoptosis as occurs in Alzheimer’s disease (15). Also, addition of estrogen to an estrogen-responsive neuroblastoma cell line modulates the mRNA levels for Nip2, a Bcl-2 interacting protein (16). This demonstrates that estrogen can regulate important mediators of apoptosis by regulating their synthesis. Although the potential for estrogen to affect gene expression patterns of apoptotic genes is large, few other examples are known.

We recently demonstrated that estrogen withdrawal induces apoptosis of cells in the chick oviduct (17). Because estrogen levels are easily modulated in the immature chick (18), this system provides an excellent model to study apoptosis in response to estrogen withdrawal. Therefore, to further elucidate the apoptotic targets of estrogen, we examined both gene expression patterns of various genes involved in apoptosis and the activation of apoptotic proteins in response to estrogen withdrawal. The data presented here demonstrate that estrogen regulates caspase activity through both transcriptional and posttranscriptional mechanisms in this hormone-responsive tissue.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RT-PCR Analysis of Various Apoptotic Genes Demonstrates that Caspase-1 and -2 Gene Expression in the Oviduct Is Stimulated by Estrogen Withdrawal
Regulation of the process leading to the commitment and execution of cells during apoptosis involves expression of genes either having proapoptotic or antiapoptotic functions. Because the withdrawal of estrogen induces apoptosis in the oviduct (17), we speculated that genes involved in one or more apoptotic pathways may be regulated by estrogen. Initially, we chose genes that are involved in different apoptotic pathways and that have been cloned in chicken (19). Bcl-2 and Bcl-XL have antiapoptotic functions and can suppress apoptosis when overexpressed (20). The heat shock protein hsp70 has been shown to suppress apoptosis by interfering with protein kinase signaling as well as by the activation of caspase-3 (21). The proto-oncogene c-myc has been implicated in determining the sensitivity of a cell to apoptosis (19). Finally, caspase-2 is involved in the early regulatory steps of the caspase proteolytic cascade (22).

Figure 1AGo is a representative set of data from multiple independent experiments comparing mRNA expression levels between an estrogen-stimulated and 5-d estrogen-withdrawn oviduct using RT-PCR. No statistically significant differences were detected in any of the gene expression patterns with the exception of caspase-2, where a consistent 4- to 5-fold up-regulation is observed in 5-d estrogen-withdrawn oviduct (Fig. 1BGo). Although the signal in the estrogen stimulated lane is difficult to see, a faint signal was detected making fold induction calculations feasible. Fold inductions were calculated using basic densitometry and are meant to be considered only semiquantitative. The same reverse transcription reactions were tested at 30 cycles to show that caspase-2 mRNA is detectable in the estrogen-stimulated oviduct, but at lower levels than that observed in the 5-d estrogen-withdrawn oviduct.



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Figure 1. Analysis of Genes Involved in Various Aspects of Apoptosis Demonstrates that Caspase-2 Is Stimulated by the Withdrawal of Estrogen in the Oviduct

A, RNA prepared from 5-d estrogen-withdrawn (W) and estrogen-stimulated (S) oviducts was subjected to RT-PCR analysis for 27 or 30 cycles with the primer pairs indicated in Table 1Go. Each primer pair was tested with RNA from at least three independent oviducts and subjected to densitometric analysis. A representative experiment is shown. B, The graph represents data generated from oviduct RT-PCR reactions at 27 cycles and is plotted normalized to actin. The error bars represent the standard deviation of three independent experiments. A 4-fold change of caspase-2 between estrogen-withdrawn and estrogen-stimulated oviduct RT-PCRs was determined to be statistically significant (P < 0.001) by Scheffé’s analysis of variance for multiple comparisons.

 
The observation that caspase-2 mRNA levels are stimulated by estrogen withdrawal prompted the examination of the other caspase genes that have been cloned from chicken. RT-PCR analysis of caspase-1, -3, and -6 (23, 24) mRNA expression levels demonstrate that caspase-1 mRNA levels are also stimulated (approximately 11-fold) by estrogen withdrawal (Fig. 2Go, A and B). Caspase-3 and -6 show no difference in mRNA expression patterns between estrogen-stimulated and estrogen-withdrawn oviducts. This demonstrates that the initiator caspases-1 and -2 are induced in response to estrogen withdrawal, whereas the executioner caspases-3 and -6 are unaffected by estrogen. Because the activation of the executioner caspases is critically dependent on the activation of the initiator caspases, this set of data suggests that the cell death pathway in oviduct may be regulated at the pretranslational as well as posttranslational level.



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Figure 2. Estrogen Withdrawal Up-Regulates both Caspase-1 and -2 mRNAs in Oviduct

A, RT-PCR analysis was conducted for 27 cycles of amplification using RNA from 5-d estrogen-withdrawn (W) and estrogen-stimulated (S) oviducts. Primer pairs for caspases-1, -2, -3, and -6 (Table 1Go) were tested with RNA from at least three independent oviducts. A representative experiment is shown. B, The bands were subjected to densitometric analysis and plotted relative to actin. The error bars represent the standard deviation of three independent experiments. The asterisk represents statistical significance for caspase-1 (P < 0.0001) and caspase-2 (P < 0.001) between the 5-d estrogen-withdrawn and estrogen-stimulated oviduct RT-PCRs.

 
Caspase-1 and -2 mRNAs Are Stimulated Within 4 h after Estrogen Withdrawal
In a previous study, we demonstrated that 1 d after estrogen withdrawal, a 6-fold increase in apoptosis is observed in oviduct (17). If an increase in caspase-1 and -2 mass is truly involved in the apoptotic response during estrogen withdrawal, then their gene expression levels should be stimulated before 1 d of estrogen withdrawal. Therefore, to determine how rapidly caspase-1 and -2 mRNA levels increase after estrogen withdrawal, RT-PCR analysis was performed on oviducts withdrawn from estrogen for various times. Figure 3AGo shows that both caspase-1 and -2 mRNA levels are fully stimulated within 1 d of estrogen withdrawal. To further define when caspase-1 and -2 mRNA levels are increased by estrogen withdrawal, RT-PCR analysis was performed on oviducts withdrawn from estrogen for times shorter than 1 d. The antiestrogen tamoxifen was injected after estrogen withdrawal to neutralize the effects of residual circulating estrogen, achieving a truly estrogen-withdrawn condition at the shorter time points. The results shown in Fig. 3Go, B–D, demonstrate that both caspase-1 and -2 mRNA levels are stimulated approximately 3-fold within 4 h of estrogen withdrawal. In Fig. 2Go, caspase-1 and caspase-2 mRNAs were shown to be up-regulated approximately 11-fold and 4.5-fold, respectively. This apparent discrepancy can be ascribed to the barely detectable amounts of caspase-1 and -2 mRNA in the estrogen-stimulated samples, making fold stimulation assessments less reliable. Nonetheless, up-regulation of caspase-1 and -2 mRNAs were consistently observed in oviducts withdrawn from estrogen for 4 h or longer.



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Figure 3. Stimulation of Caspase-1 and -2 mRNAs Occurs Within 4 h of Estrogen Withdrawal

A, Stimulated chicks were withdrawn from estrogen for 1, 2, 3, or 5 d. The oviducts were collected and subjected to RT-PCR analysis as in Fig. 1Go. Similar analysis was conducted using oviducts withdrawn from estrogen for 0.5, 1, 2, 4, 8, 12, 18, or 24 h (B and C), indicating that caspase-1 and -2 mRNAs are stimulated within 4 h following estrogen withdrawal. D, The bands from (B) and (C) were subjected to densitometric analysis and plotted relative to the estrogen-stimulated (0 h) RT-PCR. The error bars represent the SD of three independent experiments. The time points from 4–24 h of estrogen withdrawal are significantly different from the 0, 1, and 2 h time points (P < 0.01).

 
Regulation of Caspase-1 and -2 Gene Expression Occurs at the Transcriptional Level
The effects of estrogen withdrawal on expression of caspase-1 and -2 gene expression could occur either at the transcriptional level or posttranscriptional level. To address this important mechanistic question, nuclear run-on transcription assays were performed. Shown in Fig. 4Go, the transcriptional activity of both caspase-1 and -2 genes was stimulated in the 5-d estrogen-withdrawn oviduct when compared with an estrogen-treated oviduct, whereas caspase-3 and -6 levels were unchanged. Furthermore, the transcriptional activities of the caspase-1 and -2 genes were induced by 4 h of estrogen withdrawal (Fig. 4Go), which is consistent with the increase in mRNA accumulation. The estrogen-inducible transcription factor {delta}EF1 (25) is included to demonstrate the validity of the estrogen treatments and the nonestrogen-regulated transcription factor HNF3ß (26) to indicate equal cpm used for hybridization among treatments. This line of experimentation demonstrates that transcription initiation of the caspase-1 and -2 genes is occurring more frequently in estrogen withdrawal cells, suggesting that at least a portion of the rise in mRNA levels is occurring at the transcriptional level. The rapid release of repression upon estrogen withdrawal also suggests that the caspase-1 and -2 genes are direct targets of the ER. Because the increase in mRNA levels appears to be greater in the RT-PCR analysis (Fig. 2Go) compared with the nuclear run-on data, there may be additional mechanisms involved such as an increase in mRNA stability. A difference in sensitivity between the assays may also account for the discrepancy.



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Figure 4. Regulation of Caspase-1 and -2 Gene Expression Occurs at the Transcriptional Level

Nuclei isolated from oviducts either chronically stimulated with estrogen (Stim and 0 h) or withdrawn from estrogen for 1, 2, or 4 h or for 5 d (5-d W/D) were placed in nuclear run-on transcription reactions. RNA was isolated and hybridized to a filter containing cDNAs for caspases-1, -2, -3, or -6. An estrogen-stimulated gene, {delta}EF1 (25 ), and a nonestrogen-regulated gene, HNF3ß (26 ), were used as controls.

 
Caspases Become Activated in the Estrogen-Withdrawn Oviduct
The preceding data (Figs. 2–4GoGoGo) demonstrate that caspase-1 and -2 gene expression is induced by estrogen withdrawal. To determine whether the increase in caspase-1 and -2 mRNA levels leads to elevated protein levels and subsequent activation of both initiator and executioner caspases, Western blots and caspase activity assays were performed. Western blots can be used to follow the activation of a constitutively expressed caspase by monitoring the decrease of the larger proenzyme signal and/or the appearance of the smaller subunit(s) signal during apoptotic stimulation. Either of these observations suggests that the protein is being processed into the active form (24, 27). As seen in the top panel of Fig. 5AGo, the constitutively expressed executioner proenzyme of caspase-3 (32 kDa) is being processed into the active form since the procaspase signal decreases and the smaller subunit (17 and 12 kDa) signals increase in the estrogen-withdrawn cells. The intense signal detected above the capase-3 band presumably represents nonspecific interactions with the estrogen-induced ovalbumin protein, which serves as an internal control to show the cells are truly estrogen-withdrawn. Similar to caspase-3, procaspase-6 (29 kDa) decreases in the estrogen-withdrawn cells (Fig. 5AGo, middle panel), suggesting caspase-6 is also being activated. We do observe a recovery of procaspase-6 levels in the 5 d withdrawn samples, but the levels remain below fully stimulated levels. This particular caspase-6 antibody only reacts with the proenzyme form of the protein, so the smaller subunits could not be detected.



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Figure 5. Caspases Become Activated During Apoptosis of the Oviduct

Estrogen-stimulated chicks were withdrawn from estrogen for 1, 2, 3, or 5 d and total protein extracts prepared. A, Western blot analysis of estrogen-withdrawn (WD) protein extracts. Positive controls (+) used are HUT cell extracts for caspase-3 and Hela cell extracts for caspases-2 and -6. Sizes of expected, nonspecific (NS), and ovalbumin (Oval.) bands are labeled. The lower portion of the caspase-3 blot was exposed longer to visualize the smaller bands. B, Terminal deoxynucleotidyl dUTP nick end labeling analysis of estrogen-withdrawn tissues. Data are plotted as fold increase in the percentage of cells undergoing apoptosis. C, Caspase enzyme activity assays were performed on estrogen-withdrawn protein extracts using substrates for caspase-2 (valine-aspartate-valine-alanine-aspartate), caspase-3 like (aspartate-glutamate-valine-aspartate), and caspase-6 (valine- glutamate-isoleucine-aspartate). Data are plotted as the fold increase in OD405 compared with a fully estrogen-stimulated sample.

 
Because the caspase-2 gene is induced upon estrogen withdrawal (Figs. 1–4GoGoGoGo), it is not feasible to follow a decrease of the proenzyme signal when apoptosis is induced. Instead, we wanted to determine whether caspase-2 protein levels increase concomitantly with the increase in its mRNA in the estrogen-withdrawn cells and whether procaspase-2 is being activated. Figure 5AGo (bottom panel) clearly shows that procaspase-2 (45 kDa) protein levels become elevated in the estrogen-withdrawn cells, telling us that the protein increases in conjunction with its mRNA. Once again, this antibody demonstrates nonspecific cross-reactivity with the ovalbumin protein. This particular caspase-2 antibody should detect both the 45-kDa proenzyme and the 11-kDa subunit of the activated enzyme. If caspase-2 is being activated in these cells, then we had hoped that the Western blot would detect both the 45- and 11-kDa polypeptides. However, we were only able to detect the inactive proenzyme form as shown. The reasons for this may be that 1) the low levels of caspase-2 activity (see below) make detection of the activated subunit difficult under these conditions; 2) this mammalian antibody can only cross-react with the chick proenzyme form, and not the chick 11-kDa subunit; or 3) the procaspase is being degraded, not activated. This latter possibility seems improbable from the activity data in Fig. 5CGo. Unfortunately, no caspase-1 antibody is available that cross-reacts with chick caspase-1, making it impossible to examine caspase-1 through this line of experimentation. This is not too surprising because all commercial caspase-1 antibodies we found are made to mammalian forms of caspase-1, and chick caspase-1 is only approximately 40% conserved with mammalian caspase-1 (23). This set of experiments demonstrates that estrogen withdrawal leads to both an increase in protein expression levels of initiator caspases-2 and to the processing of executioner procaspase-3 and -6 molecules.

To more closely monitor apoptosis in the oviduct, terminal deoxynucleotidyl transferase dUTP nick end labeling assays were performed (17). Figure 5BGo shows that the percentage of cells undergoing apoptosis increases when estrogen stimulation is withdrawn from the oviduct compared with an estrogen-stimulated oviduct. To determine whether caspase activity corresponds with the observed increase in apoptosis, caspase activity assays were performed using estrogen-stimulated and estrogen-withdrawn oviduct whole cell lysates. These assays measure the ability of an activated caspase to cleave a colormetric marker, p-nitoaniline (pNA), from a caspase-specific substrate. The amount of freed pNA, which is relative to the levels of caspase enzyme activity, is determined by measuring the absorbance at 405 nm. The results shown in Fig. 5CGo demonstrate that the OD405 increases in the estrogen-withdrawn tissue when substrates for caspase-2 (valine-aspartate-valine-alanine-aspartate), caspase-3 like enzymes (aspartate-glutamate-valine-aspartate), and caspase-6 (valine-glutamate-isoleucine-aspartate) are used in the reaction. This suggests that caspase-2, -3 like, and -6 enzyme activity levels become elevated when the oviduct is undergoing apoptosis, albeit at different levels. These observations are consistent with other studies in which initiator caspase activity levels were generally lower than executioner caspase activity levels (28, 29, 30). The low, but consistent, levels of caspase-2 activity suggest that we were unable to detect the active enzyme on the Western blot because of insufficient sensitivity. Unfortunately, once again we were unable to assay for caspase-1 activity because of the lack of conserved sequence between chick and mammalian caspase-1. The available substrates for these assays are designed for mammalian caspases, and chick caspase-1 has several nonconserved amino acids in the substrate recognition sites compared with mammalian caspase-1 (23, 31). However, because caspase-1 appears to be regulated similarly to caspase-2, we would anticipate similar results for both the Western blot and enzyme activity assays as observed for caspase-2.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
An understanding of how estrogen modulates apoptosis is of critical importance in determining how reproductive function is regulated during reproductive cycles. It is believed that estrogen prevents apoptosis through ER-dependent mechanisms, suggesting that estrogen’s role involves gene regulation of proteins that influence apoptosis (32). Despite these observations, little is known about how estrogen actually mediates its tissue-protective effects. In the present study, we focused on the identification of estrogen-regulated gene expression and caspase activation to further understand the relationship between estrogen and apoptosis. The data presented here demonstrate that numerous apoptotic genes involved in different aspects of apoptosis are expressed in the chick oviduct. Of greater interest, caspase-1 and -2 gene expression is stimulated by estrogen withdrawal, a condition characterized by extensive apoptosis (17). This regulation occurs, at least in large part, at the transcriptional level and is only observed in the actively regressing oviduct. Furthermore, estrogen withdrawal leads to the activation of several caspases. We previously demonstrated that the BMP-7 gene is also transcriptionally stimulated by the withdrawal of estrogen and that BMP-7 protein can promote apoptosis in the oviduct (17). The data in this study therefore support the hypothesis that estrogen promotes cell survival, at least in part, by repressing the expression of genes whose protein products promote apoptosis.

The caspase family of proteins has been divided into two categories based on the length of their prodomains. Initiator caspases (-1, -2, -8, -9, -10) have long prodomains and can auto-catalyze (cleave themselves to form the active subunits) based on their ability to oligomerize via prodomain interactions (33). Executioner caspases (-3, -6, -7) have short prodomains and require other proteases, such as the initiator caspases, for processing. Our finding that caspases-1 and -2 are transcriptionally stimulated by estrogen withdrawal, whereas caspase-3 and -6 gene expression levels are not influenced by estrogen, suggests that initiator caspases are regulated differently than executioner caspases in the oviduct. We have demonstrated that apoptosis in the oviduct can be induced solely by the removal of estrogen (17). Thus, the cells apparently do not require any other external signal to activate the initiator caspases and undergo apoptosis, raising the question of how the initiator caspases become activated. One hypothesis is that, because initiator caspases can autocatalyze, simply increasing expression levels of the protein leads to activation. This idea is consistent with the induced proximity model (34), which states that bringing the initiator caspases into proximity to one another leads to auto-processing and activation. This model is supported by studies that have shown that artificially causing caspase-8 to oligomerize leads to cleavage of the proenzyme (34) and that overexpression of caspase-2 can lead to apoptosis without any additional apoptotic signal (35). We therefore propose a model in the oviduct in which the initiator procaspase molecules exists at low concentrations in the estrogen-stimulated oviduct but are not able to induce apoptosis due to a lack of activation and the presence of inhibitors of apoptosis (IAPs). Upon estrogen withdrawal, production of procaspase protein is stimulated. The increased concentrations of initiator procaspase molecules in cells leads to autocatalysis, and subsequently, an activated caspase cascade (Fig. 6Go). In any case, the data presented herein provide the first example of transcriptional regulation of caspase genes by estrogen and suggest additional means of controlling caspase activity. Whether these genes are regulated in a similar manner in other systems is a subject of continuing investigation.



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Figure 6. Model for Estrogen’s Tissue-Protective Role in Chick Oviduct

In the presence of estrogen (left panel), initiator caspase gene transcription is attenuated, leaving the executioner procaspase molecules in the inactive form. IAPs are in sufficient abundance to inhibit any active caspase. In the absence of estrogen (right panel), initiator caspase gene transcription is stimulated, resulting in more initiator caspases, which can overwhelm the steady-state levels of IAPs. This leads to the activated caspase cascade and apoptosis.

 
Most studies on caspases have focused on their posttranslational processing and activation in response to apoptotic signaling. Thus, the observation that estrogen represses the transcription of the caspase-1 and -2 genes is particularly striking. To the best of our knowledge, the only other published examples of the pretranslational regulation of caspase-1 expression by any stimulus are the withdrawal of lactogenic hormones in the mammary gland epithelium and the regression of the corpus luteum of bovine ovary (7, 36). In both of these systems, caspase-1 mRNA levels are induced concurrently with apoptosis of those cell types. These observations, in conjunction with our results, are of considerable interest because in most cases caspase-1 appears to be involved in inflammatory responses rather than in apoptosis (37). The fact that estrogen regulation of caspase-1 gene expression directly correlates with apoptosis in the oviduct, as well as in mammary gland and ovary, suggests that caspase-1 plays a critical role in apoptosis of reproductive tissues. These data may thus help resolve some of the controversy about the function of caspase-1. To the best of our knowledge, pretranslational regulation of caspase-2 has not been reported in any system, so very little is known about the regulation of this gene. Our finding that estrogen represses the transcriptional activity of the caspase-1 and -2 genes is therefore of significance in understanding the regulation of this gene family at the pretranslational level. Additional experimentation will be required to resolve the mechanisms and physiological ramifications of this regulation by estrogen.

Examination of the murine caspase-1 5'-flanking region (37) revealed that it contains a consensus sequence for NF-{kappa}B. Previous studies have implicated the squelching of NF-{kappa}B by the ER as causing repression of IL-6 gene transcription (38). The presence of an NF-{kappa}B binding site 600 bp upstream of the transcription start site of the murine caspase-1 gene suggests a mechanism whereby the withdrawal of estrogen relieves the ER inhibition of NF-{kappa}B, allowing NF-{kappa}B to activate transcription. Reduced NF-{kappa}B activity has been shown to be associated with inhibition of apoptosis by estrogen in other systems as well (39). The 5'-flanking sequence for caspase-2 has not been cloned in any species; thus, it is not possible to speculate which element(s) may be involved in its regulation by estrogen. Projects are currently underway attempting to elucidate the mechanisms by which estrogen regulates these genes.

Recent investigations into the roles of caspases-1 and -2 through the use of gene deletion experiments have provided some insight into their physiological functions. Thus far, the only phenotype reported for caspase-1 knockout mice is a defect in some of the apoptotic signaling pathways in thymocytes (40). No reproductive defects were assessed, so it is possible that defects such as the remodeling of the mammary gland upon cessation of lactation were overlooked. Our data suggest that caspase-2 has a role in apoptosis in the oviduct, but the extent of that role is unknown especially as caspase-1 may also be involved. Caspase-2 knockout mice exhibit reproductive defects characterized by the presence of excess ovarian germ cells (41), suggesting that caspase-2 is essential for the normal cell death (atresia) of primordial follicles in the fetal ovary. The stimulation of caspase-2 mRNA levels in the apoptotic estrogen-withdrawn oviduct suggests that caspase-2 plays a similar role in apoptosis in the oviduct.

In conclusion, our results describe the stimulation of caspase-1 and -2 gene transcription by estrogen withdrawal. Furthermore, the rapid stimulation of caspase-1 and -2 gene transcription after estrogen withdrawal suggests a direct role of the ER in repressing transcription of these genes. These data raise the possibility that estrogen opposes apoptosis, at least in part, by repressing initiator caspase gene transcription.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals, Hormone Treatments, and Tissue Preparation
Sexually immature, 1-wk-old White Leghorn chicks were implanted sc with pellets containing the synthetic estrogen diethylstilbestrol (DES; Hormone Pellet Press, Leawood, KA) for at least 2 wk to induce development of the oviduct. Estrogen-withdrawn chicks were produced by removal of the DES pellets for the indicated times. Acute estrogen withdrawal was performed by DES-withdrawal immediately followed by ip injection of the estrogen antagonist tamoxifen (25 mg/kg body weight) for the indicated times to neutralize the effects of the residual circulating DES. The magnum portion of the oviduct were frozen in liquid nitrogen before storage at -70 C. Total RNA was prepared as previously described (17) and stored at -70 C. All animal studies were conducted as outlined in Guidelines for Care and Use of Experimental Animals (University of Minnesota).

RT-PCR
The primers used for all RT-PCR analysis and the expected size of amplification products are listed in Table 1Go. Two micrograms of sample RNA were heated at 68 C for 15 min and reverse transcribed in 1x reaction buffer [1x First Strand Buffer, 1 mM each deoxy (d)ATP, dCTP, dGTP, and dTTP, and 1 mM oligo-deoxythymidine primer] in the presence of 10 units MMLV reverse transcriptase (Invitrogen-Life Technologies, Inc., Grand Island, NY) in a total reaction volume of 20 µl. Reactions were incubated at 42 C for 1 h followed by incubation at 68 C for 15 min. As a control for genomic DNA contamination, a RT-minus control was performed for each sample and no amplification products were detected (data not shown). Two microliters of the RT reaction were used in PCR reactions containing 1x Taq buffer, 1.5 mM MgCl2, 1 mM each dATP, dCTP, dGTP, and dTTP, 1 mM each of the specific primers, and 2.5 units Biolase DNA polymerase (Bioline, Reno, NV) in a total reaction volume of 20 µl. The PCRs were amplified for 27 cycles using the following conditions: 95 C for 30 sec, 55 C for 1 min, and 72 C for 2 min. The entire reaction (10 µl for the actin reactions) was separated by electrophoresis on a 3% Metaphor (FMC Bioproducts, Rockland, ME) agarose gel, stained in ethidium bromide, and analyzed using the Molecular Analyst software package (Bio-Rad Laboratories, Inc., Hercules, CA). Statistical significance was determined using Scheffé’s ANOVA for multiple comparisons and differences were considered significant at P < 0.05.


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Table 1. PCR Primer Sequence and Expected Fragment Sizes

 
Protein Extract Preparation and Western Blots
Total protein extracts were prepared from oviducts withdrawn from estrogen for various times by homogenization in 1x Tissue Lysis Buffer (Biosource International, Camarillo, CA) followed by centrifugation. Protein concentrations were determined using the Bradford assay kit (Bio-Rad Laboratories, Inc.). For Western blot analysis, 100–200 µg of protein extract was separated on a 4–20% Tris-HCl gradient gel (Bio-Rad Laboratories, Inc.) and transferred to nitrocellulose. The filter was blocked in 1x TBST (0.1 M Tris, 0.9% NaCl, and 0.1% Tween-20) plus 10% milk for 1 h at room temperature. Caspase-2 (sc-8985) and -3 (sc-7148) antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added to TBST in a 1:200 dilution and incubated with the filters for 1 h at room temperature. The caspase-6 antibody (StressGen, Victoria, British Columbia, Canada, AAP-106) was diluted 1:1000 in TBST. The filters were washed and incubated in a 1:20,000 dilution of goat antirabbit alkaline phosphatase-conjugated secondary antibody in 1x TBST for 1 h at room temperature. For caspase-3, the alkaline phosphatase signal was visualized using the chemiluminescent substrate Lumi-Phor (Pierce Chemical Co., Rockford, IL) and exposed to x-ray film for 3 min. For caspases-2 and -6, the alkaline phosphatase signal was detected using a NBT/BCIP solution (Pierce Chemical Co.). To assess equal loading among lanes, the blots were either stripped and reprobed with an antitubulin antibody (Santa Cruz Biotechnology, Inc.) or identical gels run and analyzed.

Caspase Assays
Caspase activity was determined from protein extracts prepared from oviducts withdrawn from estrogen for various times using the appropriate caspase assay kits (Biosource International). Briefly, 300 µg of protein extract were placed in a reaction buffer containing 200 µM caspase-specific substrate conjugated to p-nitroaniline (pNA) and incubated at 37 C for 2 h. The amount of free pNA was measured by spectrometry at 405 nm. Caspase activity corresponds to the amount of free pNA released in the reaction.

Nuclear Run-On Transcription Assays
Nuclear run-on assays were performed as previously described (42) using nuclei from oviducts withdrawn from DES (and injected with tamoxifen) for various times as described in the text. The cDNA fragments used for the genes analyzed are as follows: caspase-1 (nucleotides 40–1495) (43), caspase-2 (nucleotides 30–1308) (23), caspase-3 (nucleotides 38–1464), and caspase-6 (nucleotides 1–1673) (24). The full-length cDNA for {delta}EF1 was used as an estrogen-stimulated control (25) and the full-length cDNA for hepatocyte nuclear factor 3ß (HNF-3ß) was used as a nonestrogen-regulated control (26). The hybridized filters were exposed to x-ray film (Amersham Pharmacia Biotech, Piscataway, NJ) for 3 d at -70 C with an intensifying screen.


    ACKNOWLEDGMENTS
 


    FOOTNOTES
 
This research was supported by NIH Grants R01-DK-40082 (to M.M.S.) and TM-T32-NIDDK-0703–22 (to R.R.B.).

1 Current address: Department of Biochemistry and Molecular Biology, Mayo Graduate School of Medicine, Rochester, Minnesota 55905. Back

Abbreviations: IAPs, Inhibitors of apoptosis; pNA, p-nitoaniline.

Received for publication September 12, 2001. Accepted for publication February 5, 2002.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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