Generation of DR5-null mice.
We abolished TRAIL-DR5 signaling by targeted disruption of the extracellular cysteine-rich domain of DR5 by homologous recombination. The targeting vector replaced exons 3 and 4 and part of 5 with a neomycin resistance cassette in the antisense orientation (Fig. ). Targeted E14K ES cells were used to generate DR5-null mice. PCR on genomic DNA isolated from thymocytes confirmed the absence of the deleted sequence (Fig. ). Lack of functional gene transcription was verified by reverse transcription-PCR of RNA derived from the small bowel (Fig. ).
FIG. 1. Targeting the DR5 gene. (A) Schematic of the gene targeting strategy. Exons 3, 4, and part of 5 were targeted for deletion by homologous recombination with a vector encoding a neomycin resistance cassette. Arrowheads show the locations of forward and (more ...)
DR5-null mice show no developmental abnormalities and are fertile. We found no histological differences in the tissues examined (spleen, thymus, liver, lung, brain, intestine, colon, skin, esophagus, and kidney) from 4-week-old mice. When the organ mass of 4-to 6-week-old knockout mice was compared to that of wild-type mice, we found a statistically significant increase in the mass of the thymus in DR5-null mice (Fig. ). No particular histological compartment of the thymus appeared to be enlarged. There was no statistically significant difference in the masses of brain, heart, kidney, liver, lung, or spleen from DR5-null mice compared to wild-type mice.
Mass of the DR5 null thymus is increased compared to controls. Organs from 4- to 6-week-old mice were weighed. Statistical significance was determined by Student’s t test. *, P < 0.05.
DR5 and TRAIL are expressed in the decidua and chorion at E8.5.
To determine the function of DR5 signaling during development, we examined gene expression patterning of DR5 and TRAIL during embryogenesis by in situ hybridization. Antisense probes labeled with [35S]UTP specific for DR5 and TRAIL mRNAs were hybridized on sections of embryos at embryonic day 8.5 (E8.5), E10.5, E12.5, E13.5, E16.5, and E17.5. Sense probes for DR5 and TRAIL were used as controls. Expression of DR5 and TRAIL was close to the background in the embryo proper at all time points. However, we found DR5 expressed strongly in the vessels of the decidua and in the chorion at E8.5 (Fig. ). TRAIL expression was similar at E8.5, although the signal was weaker. TRAIL and DR5 expression was not significant in the extraembryonic tissues at other time points. Despite expression of DR5 and TRAIL in placental tissues, we have observed that DR5-null mice reproduce at a normal rate and with normal litter sizes (data not shown). Thus, the absence of TRAIL and DR5 in the placenta does not affect the reproductive process.
FIG. 3. DR5 and TRAIL are expressed at E8.5 in the placenta of wild-type mice. In situ hybridization shows staining in the decidua (d) and chorion (ch). Arrows indicate staining in the vessels of the decidua. Antisense (AS) mRNA were used on the left, and sense (more ...) Tissue-specific expression of DR5.
Previous analysis of baseline expression of murine DR5 by Northern blotting suggested that DR5 expression predominates in the heart, lung, and kidney (22
). Interestingly, Western blotting suggests that baseline expression of DR5 protein predominates in the thymus, spleen, and somewhat in the liver (Fig. ). By Western immunoblotting, tissues from DR5−/−
animals did not produce a band of the size predicted for wild-type DR5. The discrepancy between the levels of mRNA and protein suggests that posttranslational mechanisms may modulate DR5 protein levels in (some) tissues during unstressed conditions in vivo.
FIG. 4. Tissue-specific expression of DR5. DR5 protein is most prevalent in the thymus and spleen, as detected by Western immunoblotting. Weak expression of DR5 is also observed in the liver. A nonspecific band of lower molecular weight can be seen in tissue (more ...) In vitro, lack of DR5 diminishes the apoptotic response to TRAIL.
Thus far, DR5 is the only death-signaling TRAIL receptor isolated from the mouse. In order to establish whether other death-transducing TRAIL receptors exist in the mouse, we isolated DR5-null MEFs and transfected them with either E1A or empty vector. E1A transfection is associated with nuclear p53 stabilization in part through the p19ARF
). Recent reports have also uncovered decreased proteasomal degradation of p53 following E1A transfection (40
). Surprisingly, E1A transfection did not significantly alter the levels of DR5 mRNA present in MEFs (Fig. ), indicating that E1A did not increase transcription of the DR5 gene. Interestingly, E1A sensitized wild-type MEFs in a dose-dependent fashion to TRAIL, whereas DR5-null E1A MEFs remained resistant to TRAIL (Fig. ).
FIG. 5. E1A immortalization sensitizes wild-type MEFs but not DR5−/− MEFs to TRAIL. MEFs were infected with a retroviral vector expressing E1A or empty vector. (A) E1A immortalization does not significantly alter DR5 mRNA expression as determined (more ...)
We also examined the effect of adriamycin on both wild-type and DR5-null E1A MEFs. We found no significant difference in the apoptotic response to adriamycin between DR5-null and wild-type E1A MEFs (Fig. ). In order to confirm that the DR5-null MEFs lack of sensitivity to TRAIL was related to the lack of DR5 expression, we transfected murine DR5 (Killer/MK) into the DR5−/− MEFs. Following infection with a DR5-expressing retrovirus, most MEFs died during the selection process (data not shown). Surviving DR5-infected clones showed increased active caspase 3 labeling in comparison to clones containing the empty vector when challenged with TRAIL (Fig. ).
DR5-null mice are compromised in radiation-induced apoptosis.
We have previously shown that DR5 is a major target gene of p53 upregulated in response to DNA damage in the spleen, thymus, and small bowel (3
). Also, in response to ionizing radiation, p53-null mice exhibit decreased apoptosis in these tissues as well as in the colon (9
). Therefore, we hypothesized that DR5-null mice may be resistant to ionizing radiation-induced apoptosis in the spleen, thymus, small bowel, and colon. To test this idea, we exposed 4- to 6-week-old female DR5-null mice and age- and sex-matched wild-type controls to 5 Gy of gamma radiation. A separate group of DR5-null and wild-type mice were also treated with dexamethasone as a positive control. Mice were sacrificed between 6 and 8 h after irradiation, and tissues were fixed in paraformaldehyde. The time points used were known from previous studies to elicit a p53-dependent apoptotic response associated with DR5 induction in several tissues (3
Apoptosis was examined in sectioned tissues by immunohistochemistry for activated (cleaved) caspase 3 or TUNEL. Detailed analysis of the stained sections was performed by either counting the number of cells/field (activated caspase 3) or determining the area fraction that stained positive by image analysis with ImageJ software (TUNEL). For active caspase 3, single cells could be discriminated, whereas TUNEL positivity appeared mainly in the form of “apoptotic clusters” in the lymphoid organs following ionizing irradiation. Thus, the TUNEL-positive area fraction reflects the size and number of such clusters.
In the thymus, irradiated wild-type tissue showed approximately two times more apoptotic cells than DR5-null mice on average (93 ± 9 cells/field and 51 ± 21 cells/fields, respectively) as determined by counting caspase-3-positive cells (Fig. ). Approximately the same difference was observed when sections were TUNEL-stained, i.e., irradiated DR5+/+ mice had an average area fraction that was 7.03 ± 2.50 cells/field and the corresponding value for DR5−/− mice was 2.57 ± 0.25 cells/field (Fig. and ). Thus, irradiated thymus from DR5+/+ mice shows significantly increased death (P < 0.05, Student's t test) in comparison to DR5-null tissue by both active caspase 3 and TUNEL staining. Apoptosis was observed most extensively in the thymic cortex but not in the medulla in both wild-type and DR5-null mice. These results indicate that DR5 is an important but not exclusive regulator of DNA damage-induced apoptosis in the thymic cortex.
FIG. 6. Radiation-induced death is attenuated in some tissues of DR5−/− and TRAIL−/− animals. (A) DR5+/+ and DR5−/− animals were treated with 5 Gy of ionizing radiation, and tissues were harvested (more ...)
FIG. 7. Radiation-induced death is attenuated in some tissues of DR5−/− animals. DR5+/+ and DR5−/− animals were treated with 5 Gy of ionizing radiation, and tissues were harvested after 8 h. Apoptosis was detected (more ...)
FIG. 8. Image analysis of histological sections stained with TUNEL. (A) Sections from the thymus of DR5−/− animals show a smaller TUNEL-positive area fraction in comparison to thymus sections taken from DR5+/+ animals. (B) The (more ...)
In the spleen, apoptosis was strongly induced in irradiated wild-type tissues as detected by active caspase 3 and TUNEL staining (Fig. and ). Irradiated DR5-null white pulp had approximately a threefold lower apoptotic response in the white pulp on average (i.e., 182 ± 52.3 and 61.4 ± 14.4 positive cells/field for DR5+/+ and DR5−/−, respectively) of the spleen as detected by staining for active caspase 3 (Fig. ). The corresponding value for the irradiated white pulp with TUNEL-staining and image analysis was 3.7 ± 1.5 positive cells/field for DR5+/+ mice compared to 0.60 ± 0.15 DR5+/+for DR5−/− mice on average, i.e., a sixfold decrease of the TUNEL-positive area (Fig. ). Thus, analysis of caspase 3 and TUNEL-stained sections of irradiated white pulp from DR5+/+ and DR5−/− mice shows that DR5+/+ mice have significantly (P < 0.05, Student's t test) increased levels of apoptosis in the white pulp.
Irradiated red pulp showed a less than twofold increase in the number of cells with activated caspase 3 (105.4 ± 19.4 and 61.4 ± 14.4 positive cells/field for DR5+/+ and DR5−/−, respectively) (Fig. ), whereas analysis of the TUNEL-positive area fraction showed an approximately twofold increase (4.1 ± 3.0 positive cells/field for DR5+/+ animals and 1.6 ± 1.7 positive cells/field for DR5−/− animals) (Fig. ) in DR5+/+ mice compared to DR5−/− mice. Although there was a trend towards increased apoptosis in the irradiated red pulp of DR5+/+ mice, this was not statistically significant. Thus, as with the immune cells of the thymus, DR5 plays a role in the DNA damage response of the spleen and in particular in lymphocytes of the white pulp. However, since apoptotic cells are still present in both irradiated red and white pulp of DR5−/− mice, DR5 is not the sole effector of apoptosis in this tissue.
In the ileum, irradiated wild-type tissue retained a cytoplasmic active caspase 3 stain along the sides of the villi that was not evident in irradiated DR5-null ileum (Fig. ). TUNEL staining showed rare instances of apoptosis in the villi but consistent amounts of apoptosis in the crypts that was of a very slightly reduced level between the irradiated DR5-null and wild-type tissues (Fig. ). In the Peyer's patches of the ileum, the percentage of apoptotic cells was significantly greater in irradiated wild-type than DR5-null tissues (Fig. ).
In the brain, more apoptotic cells were evident by TUNEL in the white matter of irradiated wild-type compared to DR5-null mice (Fig. ). Interestingly, no radiation-induced cell death was observed in the grey matter of the DR5−/− or wild-type brain (data not shown). Since it was unclear if DR5 was upregulated in the brain of mice following irradiation, we performed comparative quantitative reverse transcription-PCR on RNA isolated from irradiated brains of wild-type and DR5−/− mice (Fig. ). By analyzing band density, we were able to document a sixfold upregulation of DR5 mRNA in the brain at 8 h following 5 Gy of whole-body irradiation (Fig. ). In order to further quantify the differences in cell death in the white matter, we counted the number of apoptotic cells per section and normalized it over the surface area of the section (Fig. ). From this analysis we conclude that there are more than twice as many TUNEL-positive cells in the wild-type white matter as in the DR5-null white matter (272 ± 126 and 104 ± 14 per mm2, respectively) following irradiation. This suggests that DR5 is critical to DNA damage-induced apoptosis in neural tissue.
FIG. 9. Radiation-induced cell death in the brains of DR5-null animals. (A) Western blot analysis shows that p53 protein levels are increased in the brain of wild-type mice (WT) 8 h following 5 Gy of gamma irradiation. Also, at this time point DR5 mRNA is upregulated (more ...)
Apoptosis was examined in other irradiated tissues, including the stomach, proximal colon, kidney, lung, and esophagus. In these tissues, the level of cell death was similar between wild-type and DR5-null mice (data not shown). In the colon, apoptosis was primarily observed basally in the crypts and became more sporadic toward the apical layer. Radiation did not induce a significant amount of cell death in the kidney, lung, or esophagus.
Dexamethasone is a steroid known to induce apoptosis in the spleen and thymus (37
). We have previously shown both in vitro (19
) and in vivo (3
) that dexamethasone treatment causes upregulation of DR5. We observed that in dexamethasone-treated mice, apoptosis was more pervasive in the thymus of wild-type mice compared to DR5-null mice (Fig. ). This difference was not observed in the spleen. These results indicate that DR5 contributes to the mechanism of dexamethasone-induced cell death in the thymus.
FIG. 10. Apoptosis in immune tissues of dexamethasone-treated animals. DR5+/+ and DR5−/− animals were injected intraperitoneally with 0.5 mg of dexamethasone/animal, and tissues were harvested after 8 h. Apoptosis was assayed by (more ...)
We hypothesized that either membrane-bound or soluble TRAIL might mediate cell death through the DR5 receptor in the lymphoid organs following irradiation. Thus, TRAIL deficiency could contribute to an attenuated apoptotic response. By performing immunofluorescence, we could confirm that TRAIL was expressed on the membrane of lymphocytes in the white pulp of the spleen (Fig. ). We also irradiated TRAIL−/− mice with 5 Gy of ionizing radiation and analyzed the amount of apoptosis in the spleen by TUNEL staining (Fig. ). There was a marked decrease in the number of TUNEL-positive cells in the white pulp of the spleen from TRAIL−/− animals analogous to that observed in the spleen of DR5−/− animals. We also observed a slight decrease in the number of apoptotic cells in the small intestine (data not shown).
FIG. 11. Cell death in the spleen of TRAIL−/− and TRAIL-treated animals. (A) Immunohistochemistry with Cy2-conjugated antibodies shows that membrane-bound TRAIL (green, middle panel) was expressed by splenocytes. Nuclei were visualized by counterstaining (more ...)
In order to confirm the role of TRAIL in promoting cell death through DR5 following DNA damage, we treated nonirradiated mice and mice irradiated with 5 Gy of ionizing radiation with soluble mouse TRAIL (50 μg/mouse injected intraperitoneally) at 4 h following the time of irradiation (Fig. ). Surprisingly, injection of TRAIL alone did not alter the number of apoptotic cells, as detected by TUNEL staining, in the spleen in comparison to the control. However, the combination treatment with TRAIL and ionizing radiation resulted in a further increase in the number of TUNEL-positive cells in the white pulp of the spleen from wild-type mice in comparison to the white pulp of DR5−/− mice at 8 h following irradiation. In some cases the white pulp of wild-type animals seemed to be completely eradicated of nonapoptotic cells. This suggests that TRAIL can modulate the level of cell death of lymphocytes in the spleen through DR5 in vivo following DNA damage.
We detected an increased number of TUNEL-positive cells in the livers of wild-type mice in comparison to the livers of DR5-deficient mice (Fig. ). TUNEL-positive cells were found primarily in the sinusoids in both wild-type and DR5-deficient mice. Kupffer cells were frequently observed in the vicinity of TUNEL-positive cells and contained TUNEL-positive particles, suggesting homing of Kupffer cells to apoptotic cells and subsequent phagocytosis. We did not find any morphological evidence for hepatocyte death in the liver, and hepatocytes were rarely TUNEL positive. We suggest that the TUNEL-positive cells found in the sinusoids of the liver may be lymphocytes based on their morphology and our previous data showing a connection between DR5 and apoptosis following irradiation in the thymus and spleen.
FIG. 12. Cell death in the liver is reduced in DR5−/− mice. Eight hours following 5 Gy of ionizing radiation, TUNEL-positive (T) cells were detected in the liver. The cells were located primarily in the sinusoids and frequently located in the vicinity (more ...)