The protein activator of RNA-activated protein kinase (PKR) is a proapoptotic protein called PACT. PKR is an interferon (IFN)-induced serine-threonine protein kinase that plays a central role in IFN's antiviral and antiproliferative activities. PKR activation in cells leads to phosphorylation of the α-subunit of the eukaryotic protein synthesis initiation factor (eIF)2α, inhibition of protein synthesis, and apoptosis. In the absence of viral infections, PKR is activated by its activator PACT, especially in response to diverse stress signals. Overexpression of PACT in cells causes enhanced sensitivity to stress-induced apoptosis. We examined PACT expression in different mouse tissues and evaluated its possible role in regulating apoptosis. PACT is expressed at high levels in colonic epithelial cells, especially as they exit the cell cycle and enter an apoptotic program. PACT expression also coincides with the presence of active PKR and phosphorylated eIF2α. These results suggest a possible role of PACT-mediated PKR activation in the regulation of epithelial cell apoptosis in mouse colon. In addition, transient overexpression of PACT in a nontransformed intestinal epithelial cell line leads to induction of apoptosis, further supporting PACT's role in inducing apoptosis.
- protein kinase
- RNA-activated protein kinase
- eukaryotic protein synthesis initiation factor 2α
interferons (IFNs) are known to have antiviral, antiproliferative, and immunomodulatory activities, which they exert by inducing several responsive genes at the transcriptional level (32). The double-straded RNA-activated protein kinase (PKR), a serine/threonine kinase, is one of the genes induced by IFNs and has been shown to be responsible for the antiproliferative and antiviral actions of IFN (20,37). Although its expression is induced by treatment with IFN, PKR is present at low constitutive levels in cells. The best-studied physiological substrate of PKR activity is the α-subunit of the eukaryotic protein synthesis initiation factor eIF2, and phosphorylation of eIF2α on Ser51 by PKR leads to inhibition of protein synthesis (31). PKR's kinase activity is exhibited only after its binding to an activator, and the most well-characterized activator of PKR is double-stranded (ds) RNA (22). Binding of an activator to PKR causes a conformational change in PKR protein, leading to an unmasking of its ATP-binding site and its autophosphorylation and activation (8). On viral infection of IFN-treated cells, PKR is activated by viral dsRNA, and this leads to a block in protein synthesis (11, 12). PKR thus plays a central role in antiviral activity of IFN. In addition, PKR is also involved in the regulation of apoptosis (10, 36), cell proliferation (14, 21), and signal transduction (38). Overexpression or activation of PKR in HeLa (13), COS-1 (33), U937 (39), and NIH/3T3 (33) cells has been shown to lead to apoptosis. Mouse embryo fibroblasts from PKR knockout mice are resistant to the apoptotic cell death in response to stress signals (3). Overexpression of PKR in a tetracycline-inducible manner and subsequent activation by dsRNA resulted in apoptosis due to expression of members of tumor necrosis factor receptor family Fas and proapoptotic Bax (1, 4).
In all of these PKR-mediated apoptotic pathways that operate in the absence of viral infections, the identity of a cellular activator of PKR remained elusive until recently. We have cloned PKR activating protein (PACT), which heterodimerizes with PKR and activates it in the absence of dsRNA (24). PACT interacts with PKR through its conserved dimerization domains, which are present in two copies in PKR (25) and in three copies in PACT (24, 27). Although the same domains are also involved in dsRNA binding, protein-protein interactions are independent of dsRNA binding as illustrated by the full dimerization activity of several dsRNA-binding defective point mutants of PKR (25, 26). Like PKR, PACT is expressed in most cell types at a very low abundance, and overexpression of PACT causes PKR activation, leading to eIF2α phosphorylation (24). In addition, PACT-overexpressing cells exhibit enhanced sensitivity to undergo apoptosis in response to serum starvation and treatments with arsenite or peroxide, indicating that these stress signals elicit signal transduction pathway(s), leading to PACT-dependent PKR activation, which results in the induction of the apoptotic cascade (9, 23). Treatment of cells with stress agents rapidly leads to phosphorylation of endogenous PACT, its association with PKR, and activation of PKR. Thus PACT has emerged as a stress-dependent activator of PKR (23).
Although we have previously shown PACT's involvement in stress-induced apoptotic pathways, its role in normal tissue homeostasis has not been examined so far. In the present study, we examined the expression pattern of the mouse homolog of PACT. Our results indicate that PACT is expressed at high levels in colonic epithelial (CE) cells and that its expression pattern suggests its role in inducing CE cell apoptosis. Furthermore, a forced overexpression of PACT in normal intestinal epithelial cells leads to induction of apoptosis.
MATERIALS AND METHODS
Subcloning and in vitro translation of mouse homolog of PACT.
The cDNA insert from the mouse expressed sequence tag (EST) clone (GenBank accession no. AA153858) was sequenced by using T7 and T3 primers initially and then by using synthetic primers from the sequenced regions. Once sequenced, the coding region was PCR-amplified and subcloned into the XbaI and BamHI sites of BSIIKS+ vector. The [35S]methionine (Perkin-Elmer)-labeled mouse PACT protein was produced by in vitro translation using the TNT in vitro translation kit (Promega).
In vitro translated, 35S-labeled human and mouse PACT proteins were synthesized by using the TNT T7-coupled reticulocyte system from Promega. Five microliters of the in vitro-translated35S-labeled proteins were incubated with 2 μl of anti-human PACT polyclonal antibody in 200 μl of IP buffer (in mM): 20 Tris · HCl, pH 7.5, 100 KCl, 1 EDTA, 1 dithiothreitol, 0.2 phenylmethanesulfonyl fluoride, and 100 U/ml aprotinin, 20% glycerol, and 1% Triton X-100 at 4°C for 30 min on a rotating wheel. Protein A-agarose (20 μl; Boehringer-Mannheim) beads were added to the mixture, and the incubation continued for an additional 1 h. The beads were washed in 500 μl of IP buffer four times, the washed beads were boiled in 2× Laemmli buffer (150 mM Tris · HCl, pH 6.8, 5% SDS, 5% β-mercaptoethanol, 20% glycerol) for 2 min, and eluted proteins were analyzed by SDS-PAGE on a 12% gel followed by phosphorimager (Storm imager, Molecular Dynamics) analysis.
Western blot analysis.
Total protein extracts (100 μg) from various organs of C57BL/6 mouse were analyzed by 12% SDS-PAGE and Western blot analysis as described before (23).
Typically, the paraffin blocks were prepared from colon of two C57BL/6 mice and each experiment was verified by using one more set of mice. That gave us data from four different mice for each antibody used. C57BL/6 mice colons were removed, flushed with cold (4°C) PBS, and opened longitudinally. Tissue samples were harvested under a dissecting microscope and immediately fixed in 10% neutral buffered formalin, paraffin embedded, and sectioned (5 μm) for immunohistochemistry. An automated Techmate 500 (Biotek) stainer was used for immunohistochemistry using the manufacturer's suggested protocol. 3,3′-Diaminobenzidine (DAB) was used as substrate for avidin biotin complex peroxidases. Antibodies used were rabbit polyclonal anti-mouse PKR (M550) antibody (1:500; Santa Cruz Biotechnology), rabbit polyclonal PACT antibody (1:1,000), rabbit polyclonal phosphospecific anti-murine PKR antibody (1:500; Biosource International), rabbit polyclonal phosphospecific anti-mouse eIF2α antibody (1:500; BioSource International), and rabbit polyclonal anti-poliferating cell nuclear antigen (PCNA) antibody (1:1,000; Santa Cruz Biotechnology). Negative control sections were incubated either with no primary antibody or with preimmune serum for PACT antibody controls. Slides were counterstained with hematoxylin, dehydrated, and mounted.
5-Bromodeoxyuridine labeling assays.
To determine mitotic turnover, two C57BL/6 mice were injected intraperitoneally with 5-bromodeoxyuridine (BrdU; 30 μg/g body wt) 1 h before death. Colonic tissues were fixed in 70% ethanol and embedded in paraffin, and 5-μm sections were prepared followed by immunohistochemistry using a Signet kit (USA-HRP detection system, murine monoclonal) with anti-BrdU (1:20) antibody. The substrate used was DAB. Slides were counterstained with hematoxylin as described in the previous section.
Identification of apoptosis.
Apoptotic cells were visualized in colon crypts using terminal deoxynucleotide transferase (TdT) labeling (TUNEL) assay, which detects apoptotic DNA strand breaks (6). In brief, colon segments were fixed in 4% paraformaldehyde and 5-μm paraffin sections were prepared by standard procedures. Sections were deparaffinized in xylene and incubated for 15 min with 20 μg/ml proteinase K (Qiagen), washed with PBS, and permeabilized (0.1% Triton X-100 in 0.1% sodium citrate and 0.01 M glycine). Endogenous peroxidase activity was blocked by incubating the sections in 0.3% H2O2 in methanol for 30 min, followed by washing in PBS. The TdT reaction mixture was prepared using an in situ cell death detection kit and fluorescein-labeled dNTPs (Roche) and was incubated in the dark at 37°C for 30 min before the reaction was terminated by washing in PBS. Each experiment was performed with a negative control (labeling solution without TdT). Fluorescein-labeled dNTPs were detected by using TUNEL POD (Boehringer-Manheim) incubated in the dark at 37°C for 30 min. Sections were washed in PBS and developed with 0.05% DAB and 0.03% H2O2 in PBS for 5 min, followed by counterstaining with hematoxylin.
The rat intestinal epithelial cell line (IEC)-6 was obtained from American Type Culture Collection and cultured in DMEM with 10% fetal bovine serum, 0.1 U/ml of bovine insulin (Sigma), and penicillin/streptomycin. The Flag-PACT/pCB6+plasmid was as described before (23, 24). Cells were cotransfected with the indicated plasmids by using the Lipofectamine (Invitrogen) reagent. Forty-eight hours after transfection, the cells were fixed in 1:1 methanol/acetone and mounted in Vectashield mounting medium containing 4,6-diamidino-2-phenylindole (DAPI). At least 300 green fluorescent protein (GFP)-positive cells were counted as alive or dead on the basis of chromatin condensation.
To study the expression pattern of the mouse homolog of PACT (24), we obtained the corresponding EST clone (GenBank accession no. AA153858) and sequenced the 1.5-kb cDNA insert. The sequence alignment of the deduced human and murine PACT proteins is shown in Fig. 1 A. As noted, murine PACT is highly homologous to PACT, differing only at eight positions. Both proteins are 313 amino acids long and identical at 305 positions. Of the eight residues that are different between the two proteins, five are conservative changes. Thus the human and mouse PACT proteins are highly homologous. Ito et al. (9) have reported cloning of RAX, which differs from mouse PACT only at two positions that could be attributed to allelic variation.
The region coding for murine PACT protein was PCR amplified from the cDNA insert and subcloned into pBSIIKS+ vector (Stratagene). We produced the murine PACT protein by in vitro translation by using this construct. As shown in Fig. 1 B, murine PACT is a 34-kDa protein and is indistinguishable by mobility from human PACT. To test further whether the polyclonal antibodies raised against the human PACT will cross-react with murine PACT, we performed immunoprecipitation analysis. As shown in Fig. 1 B, the antibodies cross-react with the murine PACT protein and can immunoprecipitate it efficiently. To confirm this, we also performed Western blot analysis on several human cell lines and one mouse cell line. As seen in Fig. 1 C, Western blot analysis detects a 34-kDa band corresponding to PACT protein in six different human cell lines and also in the murine NIH/3T3 cells. These results show that murine PACT is highly homologous to human PACT and can be detected efficiently by using the polyclonal antibodies raised against human PACT.
To study PACT expression in different mouse tissues, protein extracts were made from several different tissues, and Western blot analysis was performed. As seen in Fig. 2, Western blot analysis showed that PACT is expressed at a low level in most tissues examined and that its expression level was the highest in colon tissue. In skeletal muscle, the band corresponding to PACT was undetectable, but a faint band of slightly lower molecular weight was noted. At present, we do not know the significance, if any, of this smaller protein. To ascertain that similar quantities of protein were analyzed in all lanes, the same blot was stripped and reprobed with anti-β-actin antibody (Fig. 2, bottom). Only the spleen lane shows a somewhat higher amount of protein loading, as judged by the intensity of β-actin signal. However, because the signal for PACT is relatively weak in this lane, we concluded that PACT expression is low in the spleen. Skeletal muscle and heart lanes do not show any β-actin band, because these tissues express a different actin isoform.
Because the abundance of PACT was significantly higher in colon compared with other tissues, we then wanted to determine the cell type in colonic tissue that expressed PACT at higher levels. We therefore performed immunohistochemistry on colon sections to localize PACT expression. As seen in Fig. 3,A and B, PACT is expressed in all cell types at a very low abundance and at elevated levels in CE cells (brown staining, arrows). In particular, PACT expression was highest at the top of the crypts (arrows) and tapered off toward the base of the crypt (arrowheads). The preimmune serum control did not show any staining at the same dilution (data not shown). Because PACT is involved in stress-induced PKR activation leading to apoptosis, we sought to examine whether PKR is also expressed in CE cells. As shown in Fig.3, C and D, PKR is expressed in the CE cells. Compared with PACT expression, which was the highest at the luminal surface of epithelium and the lowest at the bases of the crypts, PKR expression pattern was relatively uniform. However, a slight increase in expression was noted at the luminal surface. CE cells are known to enter an apoptotic program as they migrate to the top of the colonic crypts (5, 34). We reasoned that because PACT levels are the highest toward the luminal surface of the colon, PKR activation might take place selectively at this surface. This would be consistent with the idea that PACT-induced PKR activation may lead to apoptosis of CE cells. To determine the presence of active, phosphorylated PKR, we stained the colon sections with antibodies specific for the phosphorylated PKR and eIF2α, which is the physiological substrate of PKR (7, 31). We used these antibodies for examining phosphorylation of eIF2α and PKR in response to stress and found them to be specific for phosphorylated proteins (23). Our immunohistochemistry analysis showed that, although PKR levels were fairly uniform throughout the colonic crypts, active or phosphorylated PKR was present only at the luminal (brown staining, arrows) surface and at the top third of the crypts of colonic crypts (Fig. 3, E and F). Consistent with this, phosphorylated eIF2α was also found to be present primarily at the luminal surface (Fig. 3, G and H; arrows) and the top one-third of the crypts. eIF2α phosphorylation has also been known to occur when cells exit the cell cycle (30). These results strongly suggest that, as the cells migrate toward the top of the crypts, they express higher levels of PACT, and this may lead either by itself or in combination with other signal(s) to activation of PKR and subsequent apoptosis. Because the proteins we are studying here express predominantly in the luminal layer of colonic epithelium, we wanted to ascertain that the immunohistochemical staining observed was not due to a nonspecific “edge-effect” staining. To confirm that the staining was specific, we did two controls. Immunohistochemical staining performed without the primary antibody, which showed no staining (Fig. 3 I; arrowheads) including the luminal edge. In addition, we performed the staining to detect expression of PCNA, which is known to localize to the proliferating cell nuclei. PCNA antibody showed no staining at the luminal epithelial layer (Fig. 3 J; arrowheads), thereby confirming that the staining obtained in Fig. 3,A–H is specific. As expected, the PCNA antibody stained the nuclei of epithelial cells in the lower one-third of the crypts, which have been shown to be actively proliferating (5). In addition to the colon, we have also examined PACT expression in the small intestine of mice. Although PACT is expressed at similar levels in small intestine and is present predominantly in epithelial cells, we do not see any gradient of expression along the villus axis (data not shown).
We extended these observations to study the proliferative status of CE cells and their apoptosis. To visualize the zone of proliferation in colonic crypts, mice were injected 1 h before death with BrdU. The BrdU antibody localized to the nuclei of proliferating cells, which were located predominantly at the base of the colonic crypts (Fig. 4 B, arrows). These results elucidate the fact that the proliferative compartment of the colonic crypts are located mainly at the lower one-third of the crypts and that the CE cells, located in the upper two-thirds of the crypt region, have exited the cell cycle. These results are in agreement with previous studies (5), which have indicated that the proliferative compartment lies in the lower one-third of the colonic crypt. We then performed TUNEL analysis on colonic tissue sections to detect for CE cells carrying fragmented DNA characteristic of apoptosing cells. As seen in Fig.4 D, these were located predominantly at the luminal surface of the colonic crypts (brown nuclear staining, arrows). Thus the cells at the luminal surface of the colon express a high level of PACT and also show presence of phosphorylated eIF2α and PKR. These same cells also stain positive for apoptosis by TUNEL assays. Proliferating cells, on the other hand, are located mainly at the lower one-third of the crypts. Our data, therefore, indicate that PACT-dependent pathways may be involved, at least in part, for the control of CE cell apoptosis. Although PACT levels seem uniformly high at the luminal layer, the apoptotic cells, as judged by TUNEL staining, are not as uniformly distributed. This indicates that, in addition to PACT-dependent PKR activation, other signals may be essential for onset of apoptotic program.
To assay for the functional significance of the higher levels of PACT in CE cells, we examined the effect of PACT overexpression on CE cell survival. We cotransfected the IEC-6 cells with a flag epitope-tagged PACT expression construct and an enhanced GFP (EGFP) expression construct. This allows us to mark and follow the apoptosis of the transfected population. Forty-eight hours after the transfection, the transfected cells were examined for hallmark signs of apoptosis, such as cell shrinkage, membrane blebbing, and nuclear condensation as seen by DAPI staining. It was observed that an overexpression of PACT resulted in pronounced chromatin condensation [Fig. 5, top, B, arrows]. To quantify the percent apoptosis within the transfected population, we counted percentage of cells showing nuclear condensation within the EGFP-positive transfected population. Only 6% of the vector-transfected cells underwent apoptosis [Fig. 5,top, D, arrowheads], whereas 21% of the PACT-overexpressing cells underwent apoptosis (Fig. 5,bottom). This indicates a 3.5-fold increase in apoptosis as a result of PACT overexpression. The expression of PACT in IEC-6 cells was confirmed by Western blot analysis with the anti-flag antibody (data not shown).
Renewal of mouse intestinal epithelium takes place rapidly and continuously (34). Proliferation in the small intestine is restricted to the mucosal invaginations termed as crypts of Liberkuhn. The stem cells at the crypt bottom give rise to four epithelial lineages, three of which differentiate as they migrate from the crypt to a villus and then upward toward the lumen of the intestine (28). Once the epithelial cells reach the upper portion of the villus, they are removed by apoptosis and extrusion (17). In the colon, there are no villi and the upward migration of epithelial cells terminates with their incorporation into a hexagonal surface epithelial cuff. In normal colon, apoptosis occurs mainly at the luminal surface, after the migration and terminal differentiation of colonocytes from the base of the crypt (17). It has been suggested that any deregulation of this normal physiological apoptosis may lead to formation and growth of colonic neoplasms.
Although the transgenic and knockout mice have provided important clues about the regulation of intestinal apoptosis (35), the exact contribution of the various apoptotic pathways remains unclear. In the normal, unstressed intestine, spontaneous apoptosis occurs at a very low level at the base of the crypt at or near the position of the epithelial stem cells. Knockout mice studies indicate that this low level of apoptosis at the base of the crypts is independent of both p53 (2, 18) and Bax (29) in both small intestine and colon. Bcl2, on the other hand, has been shown to regulate spontaneous apoptosis in colon, and Bcl2 null mice show elevated levels of apoptosis at the bases of the crypts (19). Although Bax expression has been noted at the very luminal surface in colon (15), Bax null mice show normal CE cell renewal (29).
In this report, we have examined the activation of a proapoptotic kinase, PKR, in colonic epithelium. Activation of PKR in tissue culture cells has been shown to lead to apoptosis (1, 13, 16, 23,33, 39). PACT is a protein activator of PKR, which is involved in regulating PKR activation in response to stress signals (23,24). We (23) have shown previously that treatment of cells with stress agents leads to rapid phosphorylation of PACT followed by its association with PKR, leading to activation of PKR's kinase activity. PKR activation is followed by eIF2α phosphorylation and apoptosis.
Our results, presented here, show for the first time that PACT is expressed at high levels in CE cells. It has been shown that the upper two-thirds of the colonic crypt represents epithelial cells that are no longer proliferating but continue to differentiate and migrate upward (34). By using a phosphospecific antibody for PKR and its substrate eIF2α, we have shown that the presence of activated PKR in epithelial cells coincides with the cessation of proliferation (Fig. 3,E–H). This correlates well with the immunohistochemical analysis of PACT expression, which appears to be predominantly at the upper one-third of the colonic crypts (Fig. 3,A and B) and the highest at the luminal surface. Although PKR is present in CE cells at the bottom of the crypts, presence of active PKR and phosphorylated eIF2α is detected only toward the very top of the crypts, where the levels of PACT are also higher, thereby suggesting that PACT-dependent PKR activation may occur in epithelial cells in the upper portions of the crypts. Overexpression of PACT in normal intestinal epithelial cells led to apoptosis (Fig. 5, A and B), thereby strengthening the possible role of PACT in CE cell apoptosis.
Although we observe in tissue culture cells that overexpression of PACT is sufficient to trigger an apoptotic outcome, the endogenous PACT in normal cells is rapidly phosphorylated before PKR activation by an as yet unidentified kinase in response to stress signals (23). It remains to be seen whether mere overexpression of PACT at the top one-third of the crypt may be sufficient to trigger PKR activation. Additional signals may exist that control phosphorylation of PACT and subsequent PKR activation, especially because every cell that expresses high levels of PACT does not show positive TUNEL staining. Identification of downstream pathways triggered by PKR activation or eIF2α phosphorylation and involved in mediating CE cell apoptosis may provide new clues to understanding the regulation of intestinal homeostasis.
We thank Dr. Roberd Bostick and Tonia Crooks for their help in the immunohistochemical analysis, Dr. Franklin Berger and Jody Tucker for their help in TUNEL and BrDU labeling assays, and Dr. Michael Dewey for providing the C57BL/6 mice.
This work was supported, in part, by South Carolina Cancer Center Grant E183 (to R. C. Patel).
Address for reprint requests and other correspondence: R. C. Patel, Dept. of Biological Sciences, University of South Carolina, 700 Sumter St., Columbia, SC 29208 (E-mail:).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
April 24, 2002;10.1152/ajpgi.00498.2001
- Copyright © 2002 the American Physiological Society