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BOR - Papers in Press, published online ahead of print September 15, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.030858
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BIOLOGY OF REPRODUCTION 72, 206–213 (2005)
DOI: 10.1095/biolreprod.104.030858
© 2005 by the Society for the Study of Reproduction, Inc.

The p53 Protein Influences the Sensitivity of Testicular Germ Cells to Mono-(2-Ethylhexyl) Phthalate-Induced Apoptosis by Increasing the Membrane Levels of Fas and DR5 and Decreasing the Intracellular Amount of c-FLIP1

Yamini Chandrasekaran, and John H. Richburg2

University of Texas at Austin, College of Pharmacy, Division of Pharmacology and Toxicology, Austin, Texas 78712-0125


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The consequence of mono-(2-ethylhexyl) phthalate (MEHP)-induced injury of testicular Sertoli cells is the Fas-dependent apoptotic elimination of germ cells. In addition to the well-known ability of p53 to regulate the transcription of various apoptosis-associated proteins, p53 also has been implicated in mediating the localization of Fas to the plasma membrane of various cell types in a transcription-independent manner. To resolve the role of p53 in MEHP-mediated testicular toxicity, we used wild-type (p53+/+) and p53 knockout (p53–/–) mice. A significantly lower incidence of TUNEL-positive germ cells was observed in p53–/– mice compared to p53+/+ mice at 1, 1.5, and 24 h after MEHP exposure. In these same mice, an induction of Fas and death receptor-5 (DR5) in testicular membrane preparations was observed only in p53+/+ mice. Analyses of mRNA levels in testes of p53+/+ and p53–/– mice by reverse transcription-polymerase chain reaction revealed that increases in membrane levels of Fas occurred in the absence of their transcriptional up-regulation. Processing of procaspase-8 was observed only in MEHP-treated p53+/+ mice, and this correlated with the observed incidence of germ cell apoptosis. Interestingly, the p53 status of mice also influenced the stability of c-FLIP (L), a caspase-8 inhibitory protein, that was measured at levels approximately two- to fivefold higher in p53–/– mice after MEHP-exposure compared to those in p53+/+ mice. Taken together, these data suggest that MEHP-induced germ cell apoptosis is dependent, in part, on the p53 protein and on its abilities to increase the localization of Fas and DR5 on the germ cell membrane as well as to decrease the cellular levels of c-FLIP (L).

apoptosis, Sertoli cells, signal transduction, testis, toxicology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Fas (CD95/TNFRsf6)/Fas ligand (FasL; CD95L/ TNFsf6) "death receptor" apoptotic signaling system is well described in many mammalian tissues/cells [1–3], including the testis of both rodents and humans [4–7]. Death receptor-5 (DR5) and its corresponding ligand (Tumor necrosis factor {alpha} related apoptosis-inducing ligand, TRAIL) also are expressed in the rodent testis [8, 9]. Briefly, Fas and DR5 belong to the nerve growth factor/tumor necrosis factor-receptor superfamily of surface molecules. Engagement of these receptors at the cell membrane by their cognate ligands (FasL and TRAIL, respectively) recruits the adaptor protein Fas-associated death domain protein (FADD) through protein-protein interactions at C-terminal death domains [10]. In turn, FADD recruits the proteolytic initiator caspase, procaspase-8 (FADD like interleukin 1ß converting enzyme, FLICE), via the association of shared N-terminal death effector domains, resulting in the formation of the death-inducing signaling complex (DISC) [3, 11]. The proximity of procaspase-8 molecules at the DISC allows autocatalytic processing to active caspase-8 [12]. Subsequently, caspase-8 acts on other downstream effector caspases and substrates, resulting in the characteristic cellular, biochemical, and morphological changes of apoptosis [13, 14].

In the rodent testis, participation of the Fas signaling system has been observed in response to different forms of testicular injury, including chemical injury, cryptorchidism, radiation injury, and ischemia-reperfusion [5, 6, 15–17]. Our group previously reported that Sertoli cells express FasL and initiate the apoptotic elimination of Fas-expressing germ cells after challenge with the specific Sertoli cell toxicant mono-(2-ethylhexyl) phthalate (MEHP) [5, 6]. In contrast, other investigators have reported that FasL is expressed only by mature spermatozoa and plays a role in their survival in the female genital tract [18]. However, evidence for a functional role of FasL/Fas signaling within the testis comes from studies using gld mice, which express a spontaneous point mutation in FasL (Phe 273 to Leu 273) that does not allow it to bind and activate Fas [19]. Although gld mice display apparently normal spermatogenesis, after challenge with MEHP these mice exhibit a significant reduction in the incidence of germ cell apoptosis compared to their wild-type (C57BL/6J) counterparts [9, 20]. Therefore, the significant attenuation of testicular germ cell apoptosis in gld mice indicates the functional participation of the Fas signaling system in MEHP-induced germ cell apoptosis.

A well-characterized Sertoli cell toxicant, MEHP is the active toxic metabolite of the environmental contaminant di-(2-ethylhexyl) phthalate [21–23]. The MEHP-induced disruption of Sertoli cell function results in a well-described increase in germ cell apoptosis in both young rats and mice, leading to testicular atrophy [5, 6, 9]. After MEHP-induced Sertoli cell injury, only a distinct subclass of germ cells, the primary spermatocytes and the early round spermatids, undergo apoptosis. Previous work in our laboratory has demonstrated that increased germ cell apoptosis in response to MEHP in the rat testis is preceded by increases in membrane localization of Fas [7]. However, the mechanism by which these specific subclasses of germ cells elicit this membrane expression of Fas is currently unknown.

The p53 tumor-suppressor protein influences the transcription of the genes, for Fas and DR5 in many cell types under conditions of cellular stress [24–28]. Recently, several investigators have suggested that the p53 protein also may be instrumental in the trafficking of Fas to the cell membrane from stores in the Golgi complex, in a transcription-independent manner [29, 30]. The impetus for the present work therefore was to test the involvement of p53 in modulating the sensitivity/membrane localization of Fas in germ cells. To our knowledge, MEHP has not been implicated in direct injury to germ cells; however, through injury to Sertoli cells, it creates an external environment for them that is inhospitable [22, 31]. We therefore hypothesize that MEHP-mediated Sertoli cell injury may lead to a secondary p53 response by germ cells, resulting in the increased sensitivity of germ cells to undergo apoptosis.

Here, using p53 knockout (p53–/–) mice, we demonstrate that the localization of Fas and DR5 on the germ cell membrane and the ability of the death receptors to initiate apoptosis is, in part, dependent on the expression of p53. Importantly, the changes in Fas membrane expression appear to be mediated by p53 in a transcription-independent manner. In addition, we also report the novel finding in the testis that p53 influences the cellular levels of c-FLIP (cellular FLICE-inhibitory protein), a key inhibitory protein of the Fas intracellular signaling pathway. These data suggest, to our knowledge for the first time, an interaction between the death receptor and the internal "stress-sensor" p53 protein in the regulation of testicular germ cell apoptosis after MEHP-induced Sertoli cell injury.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Approximately 3-wk-old male, C57BL/6J x 129S/v chimeric mice were obtained from the University of Texas M.D. Anderson Cancer Center (UTMDACC; Science Park Research Division, Smithville, TX). These mice were either deficient for p53 (p53–/–) or were wild-type littermates (p53+/+) and were originally created at the UTMDACC (Houston, TX) by Dr. Lawrence Donehower's research group [32]. All animals were acclimatized for 1 wk before experiments. The climate of the animal room was kept at a constant temperature (74 ± 2°F) at 35–70% humidity with a 12L: 12D photoperiod. Animals were given standard lab chow and water ad libitum. All procedures involving animals were performed in accordance with the guidelines of the University of Texas at Austin's Institutional Animal Care and Use Committee in compliance with guidelines established by the National Institutes of Health.

MEHP Exposure Protocol

To evaluate the consequences of MEHP-induced Sertoli cell injury on testicular germ cell apoptosis, 26- to 30-day-old male mice (p53+/+ and p53–/–) were given a single dose of MEHP (1 g/kg) by oral gavage, a standard procedure for the investigation of MEHP-induced testicular toxicity [33]. Both the dose and the time points used were selected based on our previous work [5–7, 9]. The MEHP (purity, >97%) was purchased from TCI America (Portland, OR). Animals received MEHP in corn oil at a volume equal to 4 ml/kg. Control animals received a similar volume of vehicle (corn oil). Vehicle and MEHP-treated animals were killed by CO2 inhalation at the time points indicated. Both testes were rapidly removed: One testis was quickly frozen in liquid N2 and stored at –80°C, and the other testis was immersion-fixed overnight in Bouin solution (Polysciences, Inc., Warrington, PA), washed in 70% ethyl alcohol-Li2CO3 (saturated solution; Mallinkrodt, Paris, KY), and embedded in paraffin.

TUNEL Analysis

Apoptotic fragmentation of DNA in mouse testis cross-sections was evaluated by TUNEL using the ApopTag kit (Intergen, Purchase, NY) and standard protocols for paraffin sections as previously reported [34]. The TUNEL-positive germ cells were quantified in each tissue section by counting the number of TUNEL-positive cells in each round seminiferous tubule. The apoptotic index reflects the percentage of seminiferous tubule cross sections with more than three TUNEL-positive germ cells. Two testicular cross-sections per mouse, with a minimum of at least 100 tubule cross-sections, were analyzed, and the data are presented as the average ± SEM from three to four mice. In the control mouse testis, the percentage of seminiferous tubules with more than three TUNEL-positive cells is approximately 4%, so an increase in apoptosis is easily determined using this method of analysis.

Western Blot Analysis

For evaluation of testicular membrane or total cellular protein expression, Western blot analysis was performed as previously described [9] using the primary antibodies against caspase-8 (AAP-118; Stressgen, Victoria, BC, Columbia), c-FLIP (06-697; Upstate Cell Signaling Systems, Charlottesville, VA), DR5 (AF837; R&D Systems, Inc., Minneapolis, MN), or Fas (sc-716; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) in three to four independent groups of mice. Equal loading was verified by either comparing expression levels of actin or, when analyzing testicular membrane protein levels, by staining the blots with amido-black.

Semiquantitative Reverse Transcription-Polymerase Chain Reaction Analysis

Total RNA was isolated from testicular tissue using the RNeasy RNA isolation kit (Qiagen, Valencia, CA). First-strand cDNA was made using 2 µg of total RNA in the presence of Superscript II reverse transcriptase and oligo-dT primer (both from Life Technologies, Carlsbad, CA). The polymerase chain reaction (PCR) was performed using 2 µl of the cDNA product and Taq DNA polymerase (Roche Applied Science, Indianapolis, IN). The PCR products of 469 base pairs (bp) for Fas, 297 bp for c-FLIP long (L), and 389 bp for actin were amplified using the following primers: Fas, 5'-catgccaacctggtaaaaaaaaagttgagg-3' (forward primer) and 5'-attggtatggtttcacgactggaggttcta-3' (reverse primer); c-FLIP (L), 5'-aatgtggactctaagcccctgcaacc-3'and 5'-cgtaggagccaggatgagtttcttcc-3'; and actin, 5'-aggcatcctgaccctgaagtac-3' and 5'-tcttcatgaggtagtctgtacg-3'. The actin mRNA was used as an internal control for the semiquantitative analysis. Conditions for the coamplification of Fas and actin were 92°C for 1 min, 59°C for 1 min, and 72°C for 35 sec for 38 cycles in 1.5 mM MgCl2, whereas that for the coamplification of c-FLIP (L) and actin were 92°C for 1 min, 58°C for 1 min, and 72°C for 40 sec for 32 cycles in 1.5 mM MgCl2.

Statistics

Significance differences between groups were evaluated using parametric single-factor ANOVA with the Fisher protected-least-significance-difference comparison with a significance value of P < 0.1 using Statview software (SAS Institute, Inc., Cary, NC).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
p53–/– Mice Have Attenuated Rates of MEHP-Induced Germ Cell Apoptosis

Analysis of the testicular histology of p53+/+ and p53–/– mice revealed similar minor aberrations in the seminiferous epithelium, including Sertoli cell vacuoles and germ cell sloughing (Fig. 1, A–D) that likely are caused by the background strain of these mice. The TUNEL analysis (Fig. 1E) showed a biphasic apoptotic response after MEHP exposure in both the p53+/+ and p53–/– mice, with an early apoptotic peak incidence at 1.5 h and a late peak incidence at 24 h after the acute dose of MEHP. The observed second peak of apoptosis matches the results of our earlier work in the C57 mouse [9]. However, tissue had not been collected in the previous study at the 1.5 h time point. The p53+/+ animals showed an approximately fourfold increase in peak apoptosis at both time points, whereas p53–/– animals displayed an approximately threefold increase with respect to their control at the same time points. A direct comparison of the apoptotic index between the p53+/+ and p53–/– mice revealed a significant reduction (~40%) in the incidence of germ cell apoptosis in the p53–/– mice at 1, 1.5, and 24 h. In both mouse strains, the specific subtypes of germ cells undergoing apoptosis are the same at all time points (Fig. 1, A–D).



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  		FIG. 1. Attenuation of germ cell apoptosis in p53–/– mice after MEHP exposure. Cross-sections of testes collected from p53+/+ (A and B) and p53–/– (C and D) mice counterstained with hematoxylin for TUNEL analysis. Sertoli cell vacuoles (asterisks) and sloughed germ cells (arrowheads) observed in the seminiferous tubules of both p53+/+ and p53–/– mice. The TUNEL-positive (brown cells) round spermatids (large arrows) and primary spermatocytes (small arrows) are indicated. The apoptotic index (E) was calculated as described in Materials and Methods. Values represent the mean ± SEM. The letter a denotes significant differences (P < 0.1) in apoptotic index between untreated and MEHP-treated mice of the same strain, and the letter b denotes significant differences (P < 0.1) between p53–/– and p53+/+ mice at matched time points. Bar = 50 µm (A and C) and 100 µm (B and D)

Membrane Death Receptor Expression Is Not Up-Regulated After MEHP in p53–/– Mice

The levels of Fas in testicular membrane preparations reached significantly elevated levels by 12 h in the p53+/+ animals after MEHP exposure (Fig. 2, A and C) and remained elevated 0.5-fold above control levels at 24 h (data not shown). In these mice, membrane DR5 levels also increased within 1.5 h and remained at elevated levels up to 12 h after MEHP exposure (Fig. 2, B and D). Increases in membrane Fas and DR5 levels were not detected above levels in untreated controls in p53–/– mice (Fig. 2). Basal levels of Fas in p53–/– mice were not significantly different from those in p53+/+ mice (Fig. 2A). These results were obtained from four independent groups of animals.



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  		FIG. 2. Western blot analysis of Fas and DR5 in membrane fractions of testicular homogenates. Representative blots from four testicular samples for each time point are provided. Shown are the Fas (~44 kDa) band intensity (A) and a graphical representation of densitometric analysis (C) in the p53+/+ and p53–/– mice exposed to MEHP. Also shown are the DR5 (~49 kDa) band intensity (B) and a graphical quantitation (D) in the p53+/+ and p53–/– mice exposed to MEHP. Values represent the mean ± SEM. The letter a denotes significant differences (P < 0.1) in membrane death receptor levels between untreated and MEHP-treated mice of the same strain, and the letter b denotes significant differences (P < 0.1) between p53–/– and p53+/+ mice at matched time points

p53 Does Not Cause Transcriptional Up-Regulation of Death Receptors in Response to MEHP

Levels of Fas mRNA were evaluated in testicular samples of animals at various time points after MEHP exposure using semiquantitative reverse transcription (RT)-PCR. After toxicant exposure, no significant changes were observed in the Fas mRNA expression in both p53+/+ and p53–/– animals (Fig. 3). The RT-PCR results for Fas at each time point are normalized with those of actin. Similar analyses of DR5 mRNA levels revealed an absence of transcription after MEHP exposure in both p53+/+ and p53–/– mice (data not shown).



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  		FIG. 3. Semiquantitative RT-PCR analysis of testicular Fas mRNA levels after MEHP exposure. Representative gels for Fas mRNA in p53+/+ (A) and p53–/– (B) mice and graphical representation of densitometric analysis (C), representing the results of three experiments, are shown. ß-Actin is used as an internal control. Values represent the mean ± SEM. No significant changes were observed

MEHP Exposure Does Not Lead to Caspase-8 Cleavage in p53–/– Animals

Procaspase-8 cleavage is a hallmark of death-receptor activation. Murine procaspase-8 is efficiently cleaved to fragments of approximately 44 kDa, approximately 31 kDa (dimer of the 18- and 10-kDa subunits), approximately 20 kDa, and approximately 10 kDa in size [35]. Here, we measured the formation of both the 44-kDa (Fig. 4) and the 31-kDa (data not shown) fragments as detected by Western blot analysis of total testicular homogenates of p53+/+ and p53–/– mice exposed to MEHP. In both mouse strains, untreated control animals expressed measurable amounts of the processed forms. A significant increase in the 44-kDa processed form of caspase-8 was measured in the p53+/+ mice at 1, 1.5, 3, and 24 h after MEHP treatment (Fig. 4). However, no significant differences with respect to untreated controls are observed in the p53–/– mice (Fig. 4).



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  		FIG. 4. Procaspase-8 processing in response to MEHP exposure. Representative blots (A) for the approximately 44-kDa processed form of caspase-8 in p53+/+ and p53–/– mice after exposure to MEHP are shown. A graphical representation (B) of the densitometric analysis for the 44-kDA form of caspase-8, normalized for actin, from four testicular samples per time point is also shown. Values are the mean ± SEM. The letter a denotes significant differences (P < 0.1) in processed caspase-8 levels between untreated and MEHP-treated mice of the same strain, and the letter b denotes significant differences (P < 0.1) between p53–/– and p53+/+ mice at matched time points

c-FLIP Protein Levels Are Increased Only in p53–/– Mice, But mRNA Levels Change in a Similar Manner in Both p53+/+ and p53–/– Mice

Western blot analysis of total testicular homogenates was carried out to analyze changes in c-FLIP (L) and short (S) after MEHP treatment. No significant changes in c-FLIP (L) and (S) levels were observed in the p53+/+ mice (Fig. 5, A and C). The opposite effect was measured in the p53–/– animals, in which c-FLIP (L) (Fig. 5, B and C) and c-FLIP (S) levels (Fig. 5B) (data not shown) were enhanced (approximately two- to fivefold) as early as 1 h after toxicant exposure. In the p53–/– mice, c-FLIP (L) levels were significantly higher than levels in the treatment controls at 1.5, 3, and 6 h after MEHP exposure, and a significant decrease between corresponding c-FLIP (L) levels in p53+/+ and p53–/– mice also was observed at these time points. Evaluation of mRNA levels of c-FLIP (L) shows similar basal levels and a similar pattern of transcriptional regulation of c-FLIP (L) in both p53+/+ and p53–/– animals following MEHP exposure (Fig. 6).



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  		FIG. 5. Western blot analysis of c-FLIP protein levels in response to MEHP exposure. Representative blots for c-FLIP (L) (55 kDa) and c-FLIP (S) (28 kDa) from p53+/+ (A) and p53–/– (B) mice exposed to MEHP, and graphical representation of densitometric analysis (C) of c-FLIP (L) levels from three experiments, normalized for actin, are shown. Values are the mean ± SEM. The letter a denotes significant differences (P < 0.1) in c-FLIP (L) levels between untreated and MEHP-treated mice of the same strain, and the letter b denotes significant differences (P < 0.1) between p53–/– and p53+/+ mice at matched time points



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  		FIG. 6. Semiquantitative analysis of c-FLIP (L) mRNA levels by RT-PCR after MEHP exposure. Representative gels for c-FLIP (L) mRNA from p53+/+ (A) and p53–/– (B) mice exposed to MEHP and a graphical representation of densitometric analysis (C), representing the results of three experiments, are shown. ß-Actin is used as an internal control. Values are the mean ± SEM. No significant changes are observed


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous work from our laboratory established the participation of the Fas/FasL signaling system in the initiation of testicular germ cell apoptosis after exposure of rodents to the Sertoli cell-toxicant MEHP [5, 6]. The FasL expressed by Sertoli cells appears to interact with Fas expressed by a specific subset of germ cells to trigger their apoptotic demise. In our previous investigations, the expression of Fas on the germ cell membrane clearly was a prerequisite for their sensitivity to apoptosis [7]. However, the mechanisms by which germ cells increase their surface expression of Fas have not been established. A novel observation of the dependence of the p53 protein in the trafficking of Fas to the cell membrane in human vascular smooth muscle cells has been described [29]. The participation of the p53 tumor-suppressor protein in the modulation of apoptosis is widely described to occur by several well-known mechanisms involving the cell-cycle protein p21 and the modulation of the transcriptional expression of various proapoptotic genes, including PUMA, NOXA, Bax, DR5, PIG3, and even Fas in some cells [24, 25, 36–39]. Therefore, in the present study, we aimed to investigate if p53-deficient mice displayed alterations in their surface membrane expression of Fas and if these mice would be less sensitive to the MEHP-stimulated loss of testicular germ cells.

The p53–/– mice used in the present study showed only minor alterations in testicular histology (Fig. 1, C and D). However, these minor changes in the seminiferous epithelium appeared to result from the background strain of these mice (C57/129S/v), because the paired wild-type mice displayed similar changes in the seminiferous epithelium (Fig. 1, A and B). Comparison of wild-type p53+/+ versus p53–/– mouse testes after MEHP exposure revealed a trend of lowered germ cell apoptosis after MEHP exposure in p53–/– mice (Fig. 1E). However, significant decreases (~40%) in apoptosis in the p53–/– mice were measured only at 1, 1.5, and 24 h. Because these time points corresponded to the two peaks of apoptosis seen in p53+/+ mice, the difference in the sensitivity of these mice was, perhaps, easier to resolve. Interestingly, the germ cell subtypes undergoing apoptosis in response to MEHP exposure were the same for both the p53+/+ and p53–/– mice (Fig. 1, A–D). In our previous studies examining the sensitivity of gld mice to MEHP-induced germ cell apoptosis, we demonstrated a significant protection from apoptosis beginning as early as 6 h after exposure, with maximal decreases (~50%) in apoptosis observed at 12 and 24 h after MEHP exposure [9, 20]. Although our present findings in the p53–/– mice do not reveal a robust protection similar to that seen in gld mice, it does reveal that p53 plays at least a partial role in modulating the MEHP-stimulated germ cell apoptosis.

Based on the findings of Bennett et al. [29] and Beltinger et al. [30], we predicted that p53–/– mice would display reduced membrane levels of Fas and not show large increases after MEHP exposure. We show that p53–/– mice display high basal membrane expression levels of Fas (though not statistically different from levels in p53+/+ mice) in the testis (Fig. 2A). In accordance with our predictions, increases in membrane Fas were observed only after MEHP exposure in p53+/+ mice (Fig. 2, A and C). These findings may explain, in part, the observed attenuation of MEHP-induced germ cell apoptosis in p53–/– mice. However, the abundant basal levels of Fas expression that are observed may account for the germ cell apoptosis that occurs.

In the present study, we show that DR5, another death receptor with an expression modulated by p53 activation, is abundantly expressed in p53–/– mice. Similar to the observations of Fas expression in p53–/– mice after MEHP exposure, DR5 membrane levels did not increase in p53–/– mice (Fig. 2, B and D). However, DR5 membrane expression was abundantly up-regulated in p53+/+ mice as early as 1.5 h after MEHP exposure and was maintained at high expression levels through 12 h. Although the expression profile of DR5 correlated very well with the first phase of caspase-8 activation as well as with germ cell apoptosis in p53+/+ mice after MEHP exposure, the ability of the DR5/ TRAIL system to initiate cell death in normal tissues is unresolved [40–42]. Future investigations therefore will be aimed at functional studies to test the ability of TRAIL to instigate germ cell apoptosis in the testis. However, our data do indicate that Fas and DR5 are expressed in p53–/– mice, although their abilities to be increased on the membrane after MEHP exposure and to increase germ cell sensitivity are severely limited.

Our initial interest in assessing the p53 protein as a likely modulator of Fas was based on the reported ability of p53 to cause membrane trafficking of Fas by a transcription-independent mechanism [29, 30]. In support of this idea, we found that Fas mRNA levels in the testis of p53+/+ mice did not significantly increase after MEHP exposure (Fig. 3, A and C). We also were unable to detect significant changes in the total Fas protein levels in the wild-type p53+/+ mice after MEHP exposure (data not shown). Therefore, the increased levels of Fas receptor in the membrane preparations of p53+/+ mice at 6 and 12 h after MEHP are not caused by the increased expression of the protein via transcription. Although the testicular membrane preparations used in our experiments are comprised of membranes of subcellular organelles as well as the plasma membrane of these cells, changes in Fas levels from Golgi and endoplasmic reticulum do not likely account for the observed changes in total membrane levels, because any increases in Fas protein in these organelle membranes reflect the production of new protein (a process that is not increased, as evidenced by the absence of significant increases in both Fas mRNA and total protein levels). Our observations that Fas levels are not increased in the testicular cell membrane preparations of p53–/– mice after MEHP exposure supports the hypothesis that functional p53 is required for Fas protein transport from Golgi or other intracellular storage sites to the membrane. Another intriguing result is the absence of DR5 transcription (data not shown) in both p53+/+ and p53–/– mice after MEHP exposure. These observations indicate the absence of active transcription by p53 in the testis, even of a robustly transcribed p53 gene DR5, after MEHP treatment.

To investigate further whether death receptors are activated in p53–/– mice, we evaluated procaspase-8 processing. Caspase-8 is autocatalytically processed from its pro-form to its active form at the DISC [43], which is created only when Fas is activated. We observed formation of the 44-kDa (Fig. 4A) and the approximately 31-kDa (data not shown) processed forms of caspase-8. The baseline levels of these processed forms are similar between the p53+/+ and p53–/– mice. However, following MEHP exposure, statistically significant increases in the level of the 44-kDa form of caspase-8 (Fig. 4B) and in the 31-kDa form (data not shown) were observed only in the p53+/+ mice, demonstrating a more robust activation of Fas in animals expressing p53. Moreover, the formation of enhanced levels of active caspase-8 that preceded the two major apoptotic peaks in wild-type mice exposed to MEHP supports our earlier results that implicated Fas activation in MEHP-mediated germ cell apoptosis. The outcome of the current experiments supports our hypothesis: Death-receptor activation in mice exposed to MEHP is p53-dependent.

The absence of p53 did not drastically reduce the basal membrane expression levels of Fas, though it may be responsible for the inability of Fas levels to increase on the membrane in response to MEHP exposure. However, these basal membrane levels of Fas could be sufficient for triggering germ cell apoptosis, which we do observe. Therefore, the attenuation of apoptosis in p53–/– mice may result from other modulators of the activity of death receptors. The Fas-activated pathway can be inhibited by the cellular FLICE inhibitory protein (c-FLIP), which contains two death effector domains and can bind FADD [44]. Mammalian cells express different splice variants of c-FLIP, of which the two main forms, c-FLIP (L) and c-FLIP (S), can inhibit death receptor-mediated apoptosis [45] by inhibiting caspase-8 activation. The c-FLIP (L) lacks a cysteine at the active site of its protease domain and inhibits apoptotic signaling by allowing only partial processing of procaspase-8, whereas c-FLIP (S), which lacks the protease domain, merely binds at the DISC to completely prevent procaspase-8 processing [44, 45]. It has been reported that c-FLIP protein can be degraded in cancer cells in a p53-dependent manner [46]. In addition, c-FLIP expression has been reported in mouse testis and been implicated in protecting the immortalized germ cell line, GC-1spg, from Fas-mediated apoptosis [47]. Therefore, we evaluated whether changes in c-FLIP protein levels in the testes occurred in both the p53+/+ and p53–/– mice following MEHP exposure and correlated with the sensitivity of germ cells to undergo apoptosis. In the p53+/+ mice, the levels of c-FLIP (L) and (S) proteins were similar and unchanged after MEHP-treatment, whereas c-FLIP (L) and (S) levels significantly increased in p53–/– mice after MEHP exposure (Fig. 5). Analysis of c-FLIP (L) mRNA levels revealed similar transcriptional profiles in response to MEHP toxicity, regardless of p53 status (Fig. 6). The absence of c-FLIP protein up-regulation in response to MEHP in p53+/+ mice, despite comparable transcription in both mouse models, suggests that c-FLIP may be degraded much more rapidly in the p53+/+ mice, as has been previously suggested [46]. This idea is also supported by the enhanced Fas:c-FLIP ratio in the p53–/– mice (data not shown). It therefore is plausible that two- to fivefold higher levels of c-FLIP in the p53–/– mice may be responsible, in part, for the observed reduced caspase-8 processing, despite their having high Fas membrane levels.

In the present study, we made the following novel observations: First, the absence of p53 expression correlates with an attenuation of germ cell apoptosis after MEHP exposure. Second, MEHP-induced increases in the membrane levels of Fas and DR5 occur only in p53+/+ mice. Third, p53 mediates increased membrane levels of Fas on germ cell membranes in response to MEHP in a transcription-independent manner. Fourth, MEHP-induced caspase-8 processing is enhanced in the testis of p53+/+ mice but not in p53–/– mice. Fifth, attenuation of MEHP-triggered germ cell apoptosis in p53–/– mice correlates with increased testicular c-FLIP protein levels. Taken together, these findings indicate that p53 may play a multitiered role in MEHP-induced germ cell apoptosis. The p53 protein not only serves to promote death-receptor membrane localization, but by bringing about the degradation of c-FLIP, p53 may act to ensure that Fas can be maximally activated in germ cells in response to MEHP-induced testicular injury.


   FOOTNOTES
 
1 Supported in part by grants from the National Institute of Environmental Health Sciences/National Institutes of Health (NIH; ES09145) and NIH Center Grant (P30 ES07784). Back

2 Correspondence: John H. Richburg, University of Texas at Austin, College of Pharmacy, PHR 5.218, 1 University Station, A1915, Austin, TX 78712-0125. FAX: 512 471 5002; john_richburg{at}mail.utexas.edu Back

Received: 9 April 2004.

First decision: 3 May 2004.

Accepted: 30 August 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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