Irod/Ian5: An Inhibitor of γ-Radiation- and Okadaic Acid-induced Apoptosis

Materials

Okadaic acid (0-8010), biszbenzimide fluorochrome (Hoechst 33342), camptothecin (C-9911), staurosporin (S-4400), daunorubicin (D-8809), doxorubicin (D1515), cycloheximide (C-6255), tumor necrosis factor (TNF)-α (T-0157), and bleomycin were from Sigma-Aldrich (St. Louis, MO). The CaMKII inhibitor KN93 was from Seikagaku America (Ijamsville, MD), and the caspase inhibitor z-val-ala-DL-asp-fluormethylketone (zVAD-fmk) was from Bachem Feinchemikal A.G. (Bubendorf, Switzerland). The transfection reagent DMRIE-C was from Invitrogen (Paisley, United Kingdom).

Irod Cloning and Modification

A human spleen cDNA library (597; http://www.rzpd.de) was screened at the Deutsche Krebsforschungs Zentrum Resource Centre (Heidelberg, Germany), by using as probe the Oar2 cDNA fragment of 419 base pairs, isolated from an OA-resistant cell clone (Sandal et al., 2001). The clone DKFZp597C0168Q3 was sequenced and found to contain a cDNA (in pSPORT1) corresponding to the full-length of a human gene (AK002158) encoding a human protein with unknown function (Irod).

For cell transfections, the Irod cDNA was excised from pSPORT1 with EcoR1/BamH1 for sense ligation and with NotI/EcoR1 for antisense ligation into pcDNA3.1/Myc-His (Invitrogen, San Diego, CA).

To visualize the localization of Irod and enable control that the whole protein sequence was present at a particular locus in the cell, the hemagglutinin (HA) epitope (YPYDVPDYA) of the influenza virus was added in-frame (5′or 3′) with Irod. These constructs were cloned into the BamH1/EcoR1 site in a modified 96J-G (pJim-GFP) retrovirus vector (Lorens et al., 2000), in-frame with green fluorescent protein (GFP), resulting in HA-Irod-GFP and Irod-HA-GFP. Truncated versions of HA-Irod were constructed with deleted GTP/GDP-binding domain (ΔGTP/GDP; deletion of amino acid 1–143) and deleted trans-membrane domain (ΔTM; deletion of amino acid 282–307).

Cell Handling and Scoring of Apoptosis

Human embryonic kidney cells (293T) were cultured in DMEM. Jurkat E-6 T lymphoma cells (American Type Culture Collection, Rockville, MD) and human prostate carcinoma cells (LNCaP; American Type Culture Collection) were grown in RPMI 1640 medium. LnCap cells stably expressing Bcl-2 (Beham et al., 1997) was a kind gift from T.J. McDonnell (The MD Anderson Cancer Center, University of Texas, Houston, TX).

All media were supplemented with 10% heat-inactivated fetal calf serum. For assays of apoptosis induction, the Jurkat cells were grown at a density of 2 × 10⁵/ml. The γ-radiation was 25 Gy by using a Cs137 γ source. 293T cells were seeded at a density of 2 × 10⁴/cm², transfected ∼18 h later, and subjected to apoptogenic stimuli ∼30 h thereafter. Apoptosis was determined as described and validated previously (Sandal et al., 2001, 2002). The background was generally no more than 5% apoptotic cells, and was subtracted from all individual calculations. Chromatin condensation was judged by microscopy of cells fixed in phosphate-buffered saline containing 2% formaldehyde and 10 μg/ml bisbenzimide (Hoechst 33342; Calbiochem, San Diego, CA).

Jurkat T-cells were transfected, using DMRIE-C, with pcDNA3.1 vector carrying Irod cDNA, antisense Irod cDNA (as-Irod), or the empty pcDNA3.1 vector. Stable transfectants were selected with G418 (400 μg/ml) for 4 wk. LNCap cells and 293T cells were transfected, using DMRIE-C and CaPO₄ precipitation, respectively, with the pcDNA3.1 constructs described above, doped (1:8) with a GFP reporter gene in pCMX-SAH/Y145F (Ogawa et al., 1995).

The 293T cells were also transfected with pBabeMN retrovirus constructs containing the 419-base pair cDNA fragment originally isolated from an OA-resistant cell line (Sandal et al., 2001), with the HA-Irod-GFP and Irod-HA-GFP in pJim-GFP vector (see above), with truncated versions of HA-Irod, coexpressed with GFP, using the IRES bicistronic retroviral vector described previously (Sandal et al., 2002), and with Bcl-2 (in pBillNeo expression vector, a gift from T.J. McDonnell). To probe the role of CaMKII 293T cells were transfected with the autocamtide-2-related inhibitory peptide (AIP; KKALRRQEAVDAL) (Ishida et al., 1995), the autocamtide-3 derived inhibitory peptide (AC3-I; KKALHRQEAVDAL) (Braun and Schulman, 1995b), as well as the inactive AC3-I derived inhibitor (AC3-C). The expression vectors (pNP220 [AIP], pNP217 [AC3-I], pNP218 [AC3-C]) were kindly provided by Dr. Ulrich Bayer and Howard Schulman (Stanford University School of Medicine, Stanford, CA). See also Fladmark et al., 2002.

Northern Blot and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis

RNA was extracted from cells by using the TRIzol Reagent (Invitrogen, Paisley, United Kingdom). For Northern blots, the RNA (20 μg/lane) was separated on a 1.2% formaldehyde/agarose gel. Irod-specific probes were produced by PCR on pcDNA3.1-Irod vector, resulting in probes specific for either full-length, 5′-end or 3′-end of the cDNA. Multiple tissue expression array (MTE) and multiple tissue Northern array (MTN; BD Biosciences Clontech) was hybridized with different Irod-specific probes according to the manufacturer's protocol.

To identify the RNA originating from the transfected DNA in Jurkat cells, cDNA was synthesized from total RNA samples using random hexamer primers and AMV-reverse transcriptase. The primary cDNA products were amplified by PCR. For detection of Irod expression, we used a T7 forward primer and an Irod-specific reverse primer (SP4): 5′-CATGCTCCATAGACCAC-3′. For detection of Irod-antisense expression a BHG reverse primer and an Irod-specific forward primer (SP5) 5′-GTTGACACGCCCTCCAT-3′. Both reactions were predicted to produce fragments of ∼1400 base pairs. T7 forward and BHG reverse primers, predicted to produce a 240-base pair fragment, were used to detect the expression of control vector.

Western Blot Analysis

Cell extraction, electrophoresis, blotting, and detection of immunoreactive proteins was as described previously (Sandal et al., 2002). The membranes were probed with 0.2% monoclonal mouse anti MDM2 (SMP14; Santa Cruz Biotechnology, Santa Cruz, CA), 0.2% monoclonal mouse anti p53 (Bp53-12; Santa Cruz Biotechnology), 1 μg/ml of polyclonal rabbit anti-caspase3/CPP32 antibody (AHZ0052; Biosource International, Nivelles, Belgium), 0.4% monoclonal mouse anti-Bcl-2 (ZS18-0193), 0.2% polyclonal rabbit anti-HA (Zymed Laboratories, South San Francisco, CA), 1 μg/ml polyclonal rabbit anti-poly(ADP-ribose) polymerase (PARP) (06-557; Upstate Biotechnology, Lace Placid, NY), or 0.1% polyclonal rabbit anti-Bax (N-20; Santa Cruz Biotechnology). Blots were reprobed with 0.01% monoclonal mouse anti β-actin (AC-15-ab6276, Abcam Limited, Cambridge, UK).

Immunofluorescence

293T cells growing on coverslips were transfected with Irod-GFP fusion constructs carrying a C- or N-terminal HA epitope (see above) and fixed 30 h thereafter. They were processed for immunofluorescence as described by Srinivasan et al., (1994), except that the cells were permeabilized with 0.1% Triton X-100, and further incubated overnight at 4°C with 1 mg/ml bovine serum albumin and 0.5% NP-40. Immunostaining was performed using mAb to the HA tag (clone12CA5; Roche Diagnostics, Indianapolis, IN), mAb to the endoplasmic reticulum marker calnexin (a gift from Dr. Ari Helenius, Swiss Federal Institute of Technology, Zurich, Switzerland), or polyclonal antibody to the Golgi marker mannosidase II (a gift from Dr. Kelley Moremen, University of Georgia, Athens, GA). The secondary antibodies (Coulter-Immunotec, Marseille, France) were tetramethylrhodamine B isothiocyanate labeled and used at 20 μg/ml. Mitochondria were visualized by staining with MitoTracker dye on live cells, according to manufacturer's protocol (Molecular Probes, Leiden, The Netherlands). Fluorescence was analyzed using a DM IRBE microscope (Leica, Heidelberg, Germany), equipped with fluorescein isothiocyanate and tetramethylrhodamine B isothiocyanate filter optics (Leica). Photomicrographs and image processing was performed using either Openlab software packages (Improvision, Coventry, United Kingdom) or Leica laser scanning confocal NT by using Leica NT software.

Bioinformatics and Statistical Analysis

The identification of putative GTP-binding domain and putative membrane binding domains of Irod was by SMART analysis (http://smart.embl-heidelberg.de/) and of putative coiled–coil regions by COILS (Lupas et al., 1991, 1996a,b; http://www.ch.embnet.org/software/COILS_form.html. Multiple sequence alignment of human (AK002158) rat (AY055776) and mouse (MacMurray et al., 2002) Irod was performed using the Clustal W multiple sequence alignment package. For determination of statistical significance the Wilcoxon paired signed rank test was used.

Irod Protects Jurkat T-Cells Specifically against Okadaic Acid- and γ-Radiation-induced Apoptosis

The previously described short cDNA fragment (Oar2) of the gene AK002158 (Irod/Ian5), which in fibroblasts protected against OA-induced death, could act by a number of mechanisms (Sandal et al., 2001). We reasoned that if Oar2 cDNA coded for a dominantly negative peptide counteracting the proapoptotic action of full-length Irod/Ian5, its action should be mimicked by transfection with antisense-Irod cDNA. If it encoded a proapoptotic peptide, its action should be mimicked by overexpression of full-length Irod. To test this experimentally we cloned AK002158 (Irod) from a human spleen cDNA library, by using Oar-2 cDNA as probe, and produced sense and antisense constructs for cell transfection. Stable expression of the transfected genes in Jurkat T-lymphoma cells was confirmed by RT-PCR (Figure 1A). The Irod cells resisted OA-induced apoptosis better than wild-type cells and cells expressing empty vector (Figure 1, B, E, and H). On the other hand, cells transfected with antisense-Irod cDNA had increased sensitivity to OA-induced death compared with wild-type cells or cells transfected with empty vector (p < 0.004 as judged by the Wilcoxon paired comparison test). This suggested that Irod itself had an antiapoptotic function.

The specificity of the antiapoptotic effect of Irod was explored using different death inducers. We first subjected the cells to γ-radiation, to which lymphoid cells are particularly sensitive (Rudner et al., 2001). The cells overexpressing Irod showed similar resistance toward apoptosis induced by γ-radiation or OA (Table 1 and Figure 1, B and C). The as-Irod–transfected cells had a significantly higher rate of apoptosis in response to γ-radiation than either wild-type cells (p < 0.005) or cells transfected with empty vector (p < 0.004). This suggested that the Jurkat cells expressed enough endogenous Irod to protect against apoptosis.

The cells overexpressing Irod were not protected against UV-C treatment (Table 1). Ionizing radiation induces double strand breaks in DNA, whereas UV-C radiation is believed to induce apoptosis mainly through single-strand DNA damage (Lu et al., 1998; Lakin and Jackson, 1999). It was therefore tested whether cells with enforced Irod expression were protected against bleomycin, which is a radiomimetic agent believed to induce apoptosis mainly via the induction of double-strand breaks in DNA (Benitez-Bribiesca and Sanchez-Suarez, 1999; Tounekti et al., 2001), or camptothecin, which is a topoisomerase inhibitor known to induce double-strand breaks (Wu et al., 2002). Irod overexpression afforded only a minor, if any, protection against any of these DNA damage-inducing agents (Table 1).

In the next series of experiments, the ability of Irod to protect against CD95 (Fas/Apo-1)-mediated apoptosis was tested. Ionizing radiation is believed to activate CD95 (Kasibhatla et al., 1998; Fujimori et al., 2000; Kasibhatla et al., 2000), and apoptosis in Jurkat cells induced by ionizing radiation and CD95-ligation is subject to common regulation downstream of caspase 8 activation (Boesen-de Cock et al., 1999). The Irod-overexpressing cells were not protected against CD95 ligation (Table 1), suggesting that Irod acted at a step in the γ-radiation pathway not shared by CD95-mediated apoptosis. The specificity of Irod was illustrated further by its inability to protect against apoptosis induced by serum withdrawal, daunorubicin, doxorubicin, or staurosporin (Table 1).

OA and γ-Radiation Induce Apoptosis through a CaMKII-dependent Pathway Inhibited by Irod, Upstream of Caspase 3 Cleavage

We have recently shown that CaMKII is activated by PP inhibitors such as OA and that CaMKII activity is required for rapid and efficient PP inhibitor-induced death (Fladmark et al., 2002). If apoptosis induced by γ-radiation and OA shared a common pathway inhibited by Irod, one might expect the CaMKII inhibitor KN93 to protect against γ-radiation as well as against OA. In fact, KN93 protected to similar extent against OA- and γ-radiation-induced apoptosis (Figure 1, B and C, and Table 1). The CaMKII inhibitor and Irod were similar also in not protecting against bleomycin, camptothecin, daunorubicin, TNF-α, staurosporin, and anti-Fas (Table 1). The addition of KN93 did not additionally protect cells overexpressing Irod against either OA (Figure 1, B, F, and I) or γ-radiation (Figure 1C). This suggested that Irod might block a CaMKII-dependent step, possibly common for the death pathway induced by γ-radiation and OA.

The degradation of the caspase substrate PARP and of procaspase 3 occurred slightly earlier in as-Irod transfectants than wild-type cells. These events were retarded in cells with enforced expression of Irod (Figure 2A). This indicated that Irod acted upstream of caspase 3 cleavage in the death pathway.

To know whether Irod affected the expression level of known apoptosis-modulating proteins, the level of p53, the proapoptotic Bax, and the antiapoptotic Bcl-2 protein was compared in wild-type cells and Jurkat cells stably transfected with Irod or as-Irod cDNA. No difference was noted in basal expression (Figure 2). The Jurkat cell p53 level was stable after irradiation. This suggested either that Jurkat cell p53, which is mutated in the C-terminal domain responsible for transactivation (Cheng and Haas, 1990), was unable to be up-regulated after MDM2 down-regulation, or that MDM2 down-regulation did not occur.

We examined next whether the ubiquitin esterase p90 human MDM2 (HDM2) was down-regulated by irradiation or OA treatment. We also considered the possibility that the antiapoptotic effect of Irod could be explained by increase of the p76 splice variant of HDM2. A high expression of HDM2 is associated with hypersensitivity to ionizing radiation (Dilla et al., 2002), and its 76-kDa splice variant, preferentially expressed in cells with high sensitivity to ionizing radiation, antagonizes p90 HDM2 (Perry et al., 2000; Evans et al., 2001). Enforced expression of Irod did not affect the resting level of either p90 or p76 HDM2. Neither did it modulate the rapid down-regulation of p90 and p76 HDM2 in response to either γ-radiation or OA (Figure 2, B and C), suggesting that Irod might act downstream of this step.

Irod Is Broadly Expressed and Can Confer OA Resistance to Cells Not Related to the Immune System

The AK002158 gene was first identified through a general sequencing effort of a human prostate cDNA library, suggesting that Irod could be expressed and have functions outside the immune system. We tested therefore whether stable enforced expression of Irod could protect also LNCaP prostate carcinoma cells against apoptosis. Irod protected the LNCaP cells better against OA-induced than daunorubicin-induced apoptosis. In contrast, LNCaP cells with stable overexpression of Bcl-2 were protected against daunorubicin-induced death and not against OA (Figure 3A). This indicated that Irod acted also on nonimmune cells, and on a death pathway that was completely different from that counteracted by Bcl-2. It could be argued that stable overexpression of Irod or Bcl-2 had led to the selection of cells with secondary properties responsible for the protection against apoptosis. To minimize this risk, we transfected 293T cells transiently with Irod cDNA, and scored them for apoptosis within 48 h after the start of transfection. Such cells were also protected selectively against OA (Figure 3B), suggesting that Irod itself was the antiapoptotic factor. Again, Bcl-2 protected against daunorubin-induced death and failed to protect against OA (Figure 3B).

The role of CaMKII activity in OA-induced 293T, cell death was established by using the cell-permeable inhibitor KN93 (for discussions of its specificity, see Fladmark et al., 2002) and expression vectors for specific peptide inhibitors of CaMKII. The specificity of these inhibitors is described by Braun and Schulman (1995a). We observed as efficient inhibition of OA-induced death in cells transfected with the autocamptide-2–related inhibitor KKALRRQEVDAL (AIP) as in cells treated with KN93 (Figure 3C). The related inhibitor KKALHRQEVDAL had similar efficiency (our unpublished data), whereas the negative control peptide (KKALDGEEAVDAL) was inefficient (Figure 3C). Cells transfected with CaMKII inhibitor were not additionally protected by Irod cotransfection (Figure 3C). The ability of Irod to protect also nonimmune cells against apoptosis prompted us to study the tissue distribution of Irod mRNA. A 1.8-kb mRNA band was detected in Jurkat T-cells and a number of human tissues, by using a probe specific for exon 3 of Irod (Figure 4A). A broad tissue distribution of Irod mRNA was revealed also by dot blot analysis (Figure 4, C and D). A probe specific for exon 2 revealed a similar size mRNA and similar tissue distribution (Sandal and Døskeland, unpublished data), suggesting a strict coexpression of exons 2 and 3. We conclude that Irod is expressed in a number of nonimmune tissues, some of which (like skeletal muscle and heart) contain mainly proliferatively quiescent long-lived cells.

Subcellular Localization of Full-Length and Truncated Irod Suggests the Presence of Separate Anchoring Domains for the Golgi/ER and Centrosomal Compartments

We questioned why Oar-2 cDNA, coding for only a small portion of Irod, could protect against apoptosis as well as full-length Irod cDNA. One explanation could be that Irod was processed in cellulo to a fragment encompassing the Oar-2 coded sequence. To know whether Irod was processed, 293T cells were transfected with cDNA for Irod with an N-terminal HA-tag and a C-terminal GFP marker. Anti-GFP or anti-HA recognized a single peptide band (70 kDa), as predicted for full-length HA-Irod-GFP (Figure 5A). Furthermore, a striking colocalization of GFP and HA was revealed at the subcellular level (Figure 5B). The HA-Irod-GFP–expressing cells were protected against OA-induced apoptosis to similar extent as cells expressing wild-type Irod (Figure 5C), suggesting that the tagged protein has full biological activity and therefore was likely to have folded correctly in the cell. We conclude that Irod is stable and can protect against apoptosis as a full-length protein.

The perinuclear localization of HA-Irod-GFP (Figure 6, B–E, left), suggested a localization of HA-Irod-GFP within centrosomal/Golgi/ER regions. In fact, we observed colocalization of Irod with the ER marker calnexin (Figure 6C), as well as with the Golgi marker mannosidase II (Figure 6D). We did not find any consistent colocalization with a mitochondria-specific dye (Figure 6E). This contrasts with the findings for HA-tagged mIan4, which localized to mitochondria when expressed in 293T cells (Daheron et al., 2001). The possibility remained that Irod could translocate to the mitochondria during apoptogenic stress, but we did not detect any such redistribution of Irod in cells treated with OA (Sandal and Døskeland, unpublished data).

Irod has a putative C-terminal transmembrane domain. Irod with this domain deleted (ΔTM-IrodΔ281–307) was mainly associated with the perinuclear, centrosomal region, and had decreased association to the ER and Golgi (Figure 7). This suggested that Irod contained a centrosomal anchoring sequence proximal to the transmembrane domain. Again, Irod behaved differently from mIan4, which assumed a diffuse cytoplasmic distribution when its C-terminal mitochondrial anchor was deleted (Daheron et al., 2001).

GTP-binding and Golgi/ER-anchoring Domains of Irod Are Dispensable for Its Antiapoptotic Effect

The 307-residue-long Irod has, in addition the hydrophobic C-terminal domain, an N-terminal highly conserved GTP-ase domain (Figure 8A). Cells expressing Irod lacking the GTP-ase domain (HA-IrodΔ1-153) or the transmembrane domain (HA-IrodΔ281-307) were protected from death as well as cells expressing wild-type Irod (Figure 8C). This indicates that residues 153–280 were responsible for the antiapoptotic action of Irod. This region is predicted to contain mainly α-helices, with coiled-coil forming potential from residues 255–280 (Figure 8). The short Oar-2 cDNA fragment (Sandal et al., 2001) was able to attenuate OA-induced apoptosis when stably transfected into prostate carcinoma cells (Figure 3A). It was also efficient when transiently transfected in 293T cells, whether controlled by a cytomegalovirus promoter (Figure 3B) or a long terminal repeat retroviral promoter (Figure 8D). The Oar-2 cDNA encodes for residues 235–285 of Irod, which include the putative coiled-coil region (Figure 8), suggesting that this region may be pivotal for the antiapoptotic effect of Irod.