INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The transcription factor nuclear factor-B (NF-B) is susceptible to activation by a variety of stimuli and conditions (Anderson et al., 1994; Koong et al., 1994; Li and Karin, 1998; Uehara et al., 1999; Gao et al., 2001). Initially, NF-B was shown to play a major role in the regulation of genes involved in inflammation and immunity (Siebenlist et al., 1994; Pomerantz and Baltimore, 1999) with more recent data indicating that it can also serve as a survival factor protecting cells from undergoing apoptosis in response to stress or toxic insult (Zhang et al., 2000; Bottero et al., 2001a). Given its significance in a number of critical biological processes, there is considerable impetus to define the factors and signaling pathways that regulate NF-B activation. The biochemical events involved in the activation of NF-B have been for the most part delineated using cytokines, primarily tumor necrosis factor- (TNF-). According to the classical paradigm, NF-B is sequestered as an inactive complex in the cytoplasm by its inhibitor, IB (Siebenlist et al., 1994). Exposure to TNF- or other stimuli results in the activation of IB kinase (IKK) (Nasuhara et al., 1999), which phosphorylates IB on two specific serine residues (Schottelius et al., 1999). The phosphorylated form of IB is then ubiquinated and degraded, unmasking the nuclear localization sequence on NF-B and allowing it to be imported into the nucleus (Duffey et al., 1999).

Among the agents capable of activating NF-B is ionizing radiation (IR). However, although there have been a number of reports of IR-induced NF-B activation in a variety of cell types, there are suggestions that the processes involved differ from those defined for TNF-. IR induces the activation of NF-B beginning 2-4 h after exposure with its activity returning to untreated levels by ~8 h (Brach et al., 1991; Mohan and Meltz, 1994; Russell et al., 2002). This is in contrast to TNF--mediated activation of NF-B, which occurs with peak activation at 30 min (Claudio et al., 1996; Raju et al., 1997; Russell et al., 2002). These disparate time courses suggest that there are fundamental differences in the TNF-- and IR-initiated signaling pathways leading to NF-B activation. Furthermore, activation of NF-B by IR was shown to occur in the absence of IB degradation in rat astrocytes and the human glioma cell line U373 (Raju et al., 1998). In addition to experimental observations comparing TNF- and IR, the obvious differences between the agents themselves (i.e., cytokine vs. physical deposition of energy) suggest the involvement of unique signaling pathways mediating the activation of NF-B.

In an attempt to define the specific processes involved in IR-induced NF-B activation, we have evaluated the events previously shown to mediate NF-B activation after TNF- treatment. Initial observations indicated that, in contrast to treatment with TNF-, IR-induced activation of NF-B in U251 cells was not associated with the degradation of IB. However, upon further investigation, the data presented herein show that IB degradation is required for IR-induced NF-B activation and can be detected when the insoluble cell fraction is evaluated. Furthermore, the IB located in the plasma membrane seems to be selectively targeted by IR, whereas TNF- degrades IB located in each of the cell fractions examined. Thus, data presented herein suggest that subcellular localization of the IB/NF-B complex can be a determining factor in the susceptibility of NF-B to IR-induced activation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Antibodies and Reagents

Antibodies to nucleoporin, calnexin, BiP, GM130, and VLA-2 were obtained from BD PharMingen (San Diego, CA). Cytochrome c oxidase I antibody was from Molecular Probes (Eugene, OR). Antibodies to IB, IB, IB, NF-B p65, epidermal growth factor receptor (EGFR), and hexokinase IV were acquired from Santa Cruz Biotechnology (Santa Cruz, CA). Dimethyl sulfoxide and human TNF- were purchased from Sigma-Aldrich (St. Louis, MO). Actin antibody was purchased from Chemicon International (Temecula, CA). MG-132 was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA).

Cell Lines and Culture Conditions

The human glioblastoma cell lines U251 and SF539 and the pancreatic BXPC-3 cell line were obtained from American Type Culture Collection (Manassas, VA) and maintained as described previously (Russell et al., 2002).

Radiation

Cultured monolayer cells were irradiated using a ¹³⁷Cs source at a dose rate of 3.7 Gy/min (U.S. Nuclear, Burbank, CA).

Electrophoretic Mobility Shift Assay (EMSA)

The preparation of nuclear extracts and EMSA analysis were described previously (Russell et al., 2002).

Separation into Insoluble and Soluble Cell Fractions

Cellular fractionation was performed as described previously (Bergo et al., 2000) with several modifications. Briefly, cell cultures (8 × 10⁶ cells) were washed twice with phosphate-buffered saline (PBS), scraped into 10 ml of PBS, and pelleted at 500 × g. The supernatant was aspirated, and the cell pellet was resuspended in three volumes of hypotonic lysis buffer (10 mM Tris, pH 7.4, 0.1 M sucrose, 10 mM NaCl, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 250 mg/ml benzamide). The suspension was incubated on ice for 10 min, subjected to three rounds of freezing/thawing, and vortexed for 30 s to ensure complete cell breakage. Samples were spun at 100,000 × g for 60 min at 4°C. The supernatant (S100) was treated with 1% NP-40 and collected as the soluble fraction and stored at 80°C. The pellet (P100) was resuspended in hypotonic lysis buffer with 1% NP-40, incubated on ice for 30 min, and vortexed for 30 s at 10-min intervals. The insoluble fraction was then stored at 80°C. Protein concentrations were determined using the DC protein assay kit (Bio-Rad, Hercules, CA).

Subcellular Fractionation

Subcellular fractionation was performed as described previously (Nantel et al., 1999) with several modifications. Briefly, cell cultures (40 × 10⁶ cells) were washed twice with PBS, scraped into 10 ml of PBS, and pelleted at 500 × g. The supernatant was aspirated, and the cell pellet was resuspended in three volumes of hypotonic lysis buffer (see above). The suspension was incubated on ice for 10 min and run three times through a 22-gauge needle to ensure complete cell breakage. Samples were then spun at 500 × g for 5 min at 4°C. The supernatant was removed and held for additional processing. The initial pellet was resuspended in 2 volumes of hypotonic lysis buffer, incubated on ice for 10 min, and spun again at 500 × g for 5 min at 4°C. The washing of the pellet was repeated twice, each time combining the extracted supernatants. Finally, the low-speed pellet (P1) was resuspended in 3 volumes of extraction buffer (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 250 mg/ml benzamide) and incubated on ice for 30 min while vortexing for 30 s at 10-min intervals. The combined supernatants were spun at 500 × g for 5 min at 4°C to remove any unbroken cells, remaining nuclei, or other large debris. The supernatant was then transferred to a new Eppendorf tube and spun at 18,000 × g for 30 min at 4°C. The resulting pellet (P2) was resuspended in two volumes of extraction buffer and incubated on ice for 30 min while vortexing for 30 s at 10-min intervals. The remaining supernatant was then spun at 100,000 × g for 60 min at 4°C. The resulting high-speed pellet (P3) was resuspended in one volume of extraction buffer and incubated on ice for 30 min while vortexing for 30 s at 10-min intervals. The supernatant (S100) was collected as the S fraction, treated with 1% NP-40, and incubated on ice for 30 min while vortexing for 30 s at 10-min intervals. Cellular fractions were then stored at 80°C and protein concentrations were subsequently determined using the DC protein assay kit (Bio-Rad).

Sucrose Density Gradients

The P2 pellets were collected as described above and subjected to sucrose density gradient centrifugation as described in Hubbard et al. (1983) and Meier et al. (1984) with several modifications. Sucrose density gradients were generated by layering three concentrations of sucrose (1.5, 0.75, and 0.375 M; chilled to 4°C) in 3-ml volumes in an ultracentrifuge tube. Samples (P2 pellets) were resuspended in 1 ml of 0.375 M sucrose and added to the top layer. The gradients were then spun at 100,000 × g for 4 h at 4°C. Tubes were then removed and kept on ice. The interface between sucrose layers was collected by inserting an 18-gauge needle into the interface and removing a volume of 1.5 ml. The interface between the 1.5 and 0.75 M sucrose was collected as the lower interface and the interface between the 0.75 and 0.375 M sucrose was collected as the upper interface. The extracted volumes were then resuspended to 3 ml 0.375 sucrose and spun at 100,000 × g for 1 h at 4°C. The supernatant was aspirated and the pellet was resuspended in 1 volume of extraction buffer. Sucrose gradient cell fractions were then stored at 80°C. Protein concentrations were subsequently determined using the DC protein assay kit (Bio-Rad).

Plasma Membrane Isolation

Isolation of a pure plasma membrane fraction is limited by contamination with endoplasmic reticulum (ER) (Hubbard et al., 1983). Lin et al. (1988) describe a method of adding CaCl₂ to cell lysates to reduce ER contamination and enrich plasma membrane fractions. Direct isolation of the plasma membrane (PM) was performed as described previously (Lin et al., 1988) with several modifications. Isolation of the P2 pellet was performed as stated above; however, for these studies the pellet was resuspended in 400 µl of hypotonic lysis buffer with CaCl₂ added to a final concentration of 10 mM. After incubation on ice for 10 min, the sample was gently vortexed and diluted to 1 ml with 0.375 M sucrose. The CaCl₂-treated sample was then subjected to a sucrose density gradient as described above. The lower interface was then isolated, spun down at 100,000 × g for 1 h at 4°C, and resuspended in 1 volume of extraction buffer. The PM fraction was then stored at 80°C. Protein concentrations were subsequently determined using the DC protein assay kit (Bio-Rad).

Endoplasmic Reticulum Isolation

Isolation of the ER was achieved as follows. Briefly, cell cultures (40 × 10⁶ cells) were washed twice with PBS, scraped into 10 ml of PBS, and pelleted at 500 × g. The supernatant was aspirated and the cell pellet was resuspended in 3 volumes of hypotonic lysis buffer (see above). The suspension was incubated on ice for 10 min and run three times through a 22-gauge needle to ensure complete cell breakage. Samples were then spun at 500 × g for 5 min at 4°C. The initial supernatant was removed and discarded. The remaining nuclear pellet was resuspended in 2 volumes of hypotonic lysis buffer and spun at 500 × g for 5 min at 4°C. The washing of the pellet was repeated three times. The nuclear washes were then combined and were spun at 500 × g for 5 min at 4°C to remove any unbroken cells, remaining nuclei, or other large debris. The supernatant was then diluted to a volume of 1 ml with 0.375 M sucrose. The sample was then subjected to a sucrose density gradient as described above. The lower interface was isolated, spun at 100,000 × g for 1 h at 4°C, and resuspended in 1 volume of extraction buffer. The ER fraction was then stored at 80°C. Protein concentrations were subsequently determined using the DC protein assay kit (Bio-Rad).

Immunoblot Analysis

Immunoblot analysis was performed as described previously (Russell et al., 2002).

Confocal Microscopy

Confocal microscopy was performed as described previously (Pepin et al., 1994) with several modifications. Briefly, 1 × 10⁵ cells were plated in four-well chamber slides and allowed to grow for 48 h. Slides were then treated with either radiation (10 Gy) or TNF- (10 ng/ml). Immediately after treatment the media were replaced with serum-free media containing the EGFR antibody at a 1:50 dilution for 2 h. At the appropriate time point (2 h after IR and 30 min after TNF), the slides were fixed in 4% paraformaldehyde for 2 min and permeabilized for 3 min in a 1% NP-40/PBS solution. Slides were then washed and allowed to incubate with the IB antibody at a 1:50 dilution in 1% bovine serum albumin in PBS for 2 h. After washing with PBS, the appropriate secondary antibodies (fluorescein isothiocyanate and Texas Red conjugates) were applied at a dilution of 1:100 in 1% bovine serum albumin for 1 h. Slides were then washed, the chambers removed, and antifade (DAKO, Carpinteria, CA) added to the slide. Coverslips were then applied and sealed with nail polish. Slides were then observed under a laser scanning 510 LSM confocal microscope (Carl Zeiss, Thornwood, NY). Images were captured using LSM software package (Carl Zeiss).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

As an initial characterization of radiation-induced NF-B activation in the human glioma cell line U251, NF-B/DNA binding was determined as a function of time after exposure to 10 Gy. As shown in Figure 1, there is a relatively low basal level of NF-B activity in U251 cells, which is increased by IR in a time-dependent manner, reaching a maximum at 2 h and returning to control levels by 8 h after exposure. This time course is in contrast to U251 cells treated with TNF-, in which case maximal NF-B activity was induced within 30 min (Russell et al., 2002).

View larger version (40K): [in this window] [in a new window]   Figure 1.   IR-induced NF-B activation. Cells were irradiated (10 Gy) and collected at the specified times for EMSA analysis. Asterisk (*) indicates lane containing a 100-fold excess of cold NF-B oligonucleotide competitor. C, unirradiated control sample.

According to the classical paradigm, a critical event in NF-B activation is the degradation of IB, which allows NF-B to translocate to the nucleus (Karin and Ben Neriah, 2000). Thus, as a further comparison between TNF- and IR-induced NF-B activation, IB protein levels were determined in U251 whole cell lysates after each treatment. In U251 cells treated with TNF-, IB levels were significantly reduced by 5 min and returned to control levels by 60 min (Figure 2A), consistent with previous reports (Claudio et al., 1996). In contrast, after exposure to IR, no significant degradation of IB was detected (Figure 2B). In addition to IB, other IB proteins have been shown to play a role in the regulation of NF-B activation (Baeuerle and Baltimore, 1996; Renard et al., 2000). To determine whether IB and IB were involved in IR-induced NF-B in U251 cells, the levels of these two proteins were determined by immunoblot analysis. As shown in Figure 3A, TNF- exposure resulted in the degradation of IB with the same kinetics as IB, but did not affect IB. Treatment with IR had no effect on levels of IB or IB (Figure 3B).

View larger version (39K): [in this window] [in a new window]   Figure 2.   IB protein levels in U251 cells after exposure to TNF- or IR. Cells were treated with TNF- (10 ng/ml) (A) or irradiated (10 Gy) (B) and collected at the specified times for immunoblot analysis. Actin was used as a loading control. 0, untreated control sample.

View larger version (49K): [in this window] [in a new window]   Figure 3.   IB and IB protein levels in U251 cells after exposure to TNF- or IR. Cells were treated with TNF- (10 ng/ml) (A) or irradiated (10 Gy) (B) and collected at the specified times for immunoblot analysis. Actin was used as a loading control. 0, untreated control sample.

The above-mentioned results suggested that degradation of IB is not necessary for radiation-induced NF-B activation in U251 cells. To test this hypothesis, U251 cells were treated with the proteosome inhibitor MG-132. If IB degradation is not required for NF-B activation in U251 cells, MG-132 should not affect NF-B DNA binding after IR. However, as shown in Figure 4, MG-132 significantly reduced radiation-induced NF-B DNA binding activity, indicating that degradation of IB is indeed an essential step in IR-induced NF-B activation.

View larger version (18K): [in this window] [in a new window]   Figure 4.   Effect of MG-132 on IR-induced NF-B activation. Cells were treated with MG132 (50 µM, 2 h) or dimethyl sulfoxide (vehicle control), irradiated (10 Gy), and collected 2 h later for EMSA analysis. Asterisk (*) indicates lane containing a 100-fold excess of cold NF-B oligonucleotide competitor.

Because IB exists primarily in the cytoplasm, its degradation after exposure to activators of NF-B is typically evaluated and detected using whole cell lysates, as shown in Figure 2, A and B. However, IB has also been detected in other subcellular compartments (Suyang et al., 1996; Hay et al., 1999; Rodriguez et al., 1999; Sachdev et al., 2000). To determine whether radiation affects IB located at other sites, U251 cell lysates were separated into soluble (S) and insoluble (I) fractions (see MATERIALS AND METHODS) and IB levels determined by immunoblot analysis. For these studies, cytochrome c oxidase I, which is localized to the mitochondrial membrane, was used as a marker for the insoluble fraction and hexokinase IV, which is found in the cytoplasm, was used as a marker for the soluble fraction (Figure 5). In U251 cells the majority of IB is present in the soluble fraction, with a significantly smaller level found in the insoluble fraction. Whereas treatment of U251 cells with TNF- induced degradation of IB in both fractions, exposure of cells to IR had no effect on IB levels in the soluble fraction, which corresponds to the cytoplasm, but did result in the loss of IB from the insoluble fraction. To examine the effects of IR and TNF- on the other isoforms of IB, additional immunoblot analysis was performed on the insoluble and soluble fractions. Although IB was found in both insoluble and soluble fractions, IB was only detected in the soluble fraction. Whereas the soluble pool of IB was reduced after TNF- treatment, IR had no effect on IB or IB levels.

View larger version (33K): [in this window] [in a new window]   Figure 5.   IB, IB, and IB protein levels in insoluble and soluble fractions of U251 cells after exposure to IR or TNF-. Cells were mock irradiated, irradiated (10 Gy, 2 h), or treated with TNF- (10 ng/ml, 30 min) and collected for immunoblot analysis. Cytochrome c oxidase I and hexokinase IV were used as markers of the insoluble and soluble fractions, respectively.

The effects of IR on IB located in the insoluble fraction were then characterized as a function of time after irradiation and dose (Figure 6). After 10 Gy a decrease in IB levels in the insoluble fraction was detected by 1 h and remained suppressed until 24 h post-IR. No significant change in IB levels was detected in the soluble fraction at any of the time points examined after IR. At 2 h after IR, doses as low as 5 Gy resulted in loss of IB from the insoluble fraction, whereas doses of up to 40 Gy had no significant effect on IB in the soluble fraction. The loss of IB from the insoluble fraction is consistent with IR-induced NF-B activation in terms of dose and time course. Furthermore, these data account for the failure to detect IB degradation after IR when whole cell lysates are evaluated. That is, the IB located in the soluble fraction (cytoplasm), which is not affected by IR, masks the IR-induced loss of IB in the insoluble fraction, which is present in considerably smaller levels.

View larger version (36K): [in this window] [in a new window]   Figure 6.   IB protein levels in the insoluble and soluble fractions of irradiated U251 cells. (A) Cells were treated with IR (10 Gy), collected at the specified times, separated into insoluble and soluble fractions, and subjected to immunoblot analysis. (B) Cells were treated with the indicated doses of IR, collected after 2 h, separated into insoluble and soluble fractions, and subjected to immunoblot analysis. Cytochrome c oxidase I and hexokinase IV were used as markers of the insoluble and soluble fractions, respectively. C, mock-irradiated control sample.

To determine whether the existence of IR-sensitive IB in the insoluble fraction was unique to U251 cells, two additional cell lines were evaluated. Previously, we showed that the SF539 glioma cell line had a low basal level of NF-B activity, which was significantly increased after 10 Gy IR with the same kinetics as the U251 cell line (Russell et al., 2002). In contrast, the BXPC-3 pancreatic cell line has a relatively high basal level of NF-B activity that is not enhanced by IR (Russell et al., 2002). Evaluation of immunoblots of whole cell lysates from SF539 and BXPC-3 cell line did not show any degradation of IB after 10 Gy IR (our unpublished data). As shown in Figure 7A, when SF539 cell lysates were separated into insoluble and soluble fractions, IB is detectable in both fractions; IR induced the loss of IB only from the insoluble fraction, whereas treatment with TNF- resulted in degradation of IB located in the insoluble and soluble fractions. These results are similar to those for U251. In contrast, in the BXPC-3 cell line, IB was only detected in the soluble fraction, which was reduced after TNF- treatment but not after IR (Figure 7B). The same markers (cytochrome c oxidase I and hexokinase IV) used for U251 cells described above were used as a measure of the effectiveness of the isolation procedure in these cell lines.

View larger version (47K): [in this window] [in a new window]   Figure 7.   IB protein levels in insoluble and soluble fractions of SF539 and BXPC-3 cells after exposure to IR or TNF-. (A) SF539 cells were mock irradiated, irradiated (10 Gy, 2 h), or treated with TNF- (10 ng/ml, 30 min), collected, and separated into insoluble and soluble fractions, and subjected to immunoblot analysis. (B) BXPC-3 cells were mock irradiated, irradiated (10 Gy, 2 h), or treated with TNF- (10 ng/ml, 30 min), collected, separated into insoluble and soluble fractions, and subjected to immunoblot analysis. Cytochrome c oxidase I and hexokinase IV were used as markers of the insoluble and soluble compartments, respectively.

As a first step in determining the specific subcellular site(s) in which the IR-sensitive IB is located, U251 cell lysates were subjected to serial differential centrifugation (see MATERIALS AND METHODS). Cell lysates were spun at specified rates, which resulted in four fractions (P1, 500 × g pellet; P2, 18,000 × g pellet; P3, 100,000 × g pellet; S, 100,000 × g supernatant) and subjected to immunoblot analysis. As shown in Figure 8A, IB was found in all three pellets representative of the insoluble fraction (P1, P2, and P3), as well as in the S fraction, which corresponds to the cytoplasm. Whereas TNF- degraded IB in all four fractions (Figure 8A), IR induced the loss of IB only from the P2 fraction (Figure 8B). The lack of effect of IR on the pool of IB localized to the S fraction as well as the IR-induced degradation of IB in an insoluble fraction (P2) was consistent with previous results in this study (Figure 5). To define the organelles found in the P2 fraction, immunoblots were probed with antibodies to specific protein markers corresponding to the nucleus (nucleoporin), mitochondria (cytochrome c oxidase I), plasma membrane (VLA-2), ER (BiP), Golgi (GM130), and cytoplasm (hexokinase IV). As shown in Figure 8C, the P2 pellet, which contained the IR-sensitive IB, was composed of mitochondria, plasma membrane, ER, and Golgi complex and was relatively free of nuclei or cytoplasm. None of the organelle markers were affected by treatment with IR.

View larger version (21K): [in this window] [in a new window]   Figure 8.   IB protein levels in subcellular fractions of U251 cells after exposure to TNF- or IR. (A) Cells were treated with TNF- (10 ng/ml, 30 min) or (B) irradiated (10 Gy, 2 h), collected, separated into specified subcellular fractions (see MATERIALS AND METHODS), and subjected to immunoblot analysis. Actin was used as a loading control within a particular subcellular fraction. (C) Identification of organelles localized to P2 fraction of U251 cells. Cells were mock irradiated or irradiated with 10 Gy, collected at 2 h, separated into specified subcellular fractions, and subjected to immunoblot analysis for protein markers of cellular organelles. W, whole cell lysate, which was used as a positive control.

To delineate the components of the P2 fraction isolated from U251 cells, the P2 pellet was added to a sucrose density gradient (SDG) and subjected to high-speed centrifugation (see MATERIALS AND METHODS). Immunoblot analyses of the SDG interfaces were then performed. As shown in Figure 9A, IB and p65 NF-B were both localized to the lower interface fraction of the SDG. When the same procedure was performed on U251 cells exposed to 10 Gy 2 h previously there was a significant loss of each protein, which is consistent with IB being degraded and NF-B translocating to the nucleus (Karin and Ben Neriah, 2000). To identify the subcellular components in the lower interface of the SDG, the same marker proteins used in Figure 8C were analyzed. As shown in Figure 9B, the lower interface of the SDG, which contained the IR-sensitive IB, was enriched in plasma membrane and ER and relatively free of mitochondria or Golgi complexes.

View larger version (36K): [in this window] [in a new window]   Figure 9.   IB and p65 NF-B protein levels after sucrose density gradient separation of the P2 fraction of U251 cells. (A) Cells were mock irradiated or irradiated with 10 Gy, collected at 2 h, separated into specified subcellular fractions, and subjected to a sucrose density gradient (see MATERIALS AND METHODS). Immunoblot analysis was performed on the resulting protein pellet. Actin was used as a loading control within a particular subcellular fraction. (B) Marker proteins corresponding to specific organelles after the P2 fraction was subjected to sucrose density gradient separation. W, whole cell lysate, which served as a positive control.

The results from Figure 9 suggest that the radiosensitive pool of IB is associated with either the ER or PM. To separate ER from the PM it was necessary to use individual isolation schema. For the PM fraction, CaCl₂-treated P2 pellets (Lin et al., 1988) were passed through the SDG. The lower interface of the SDG was then isolated and subjected to immunoblot analyses. Enriched ER fractions were obtained from combined washes of the low-speed (nuclear) pellet followed by SDG separation. The lower interface of the SDG was then isolated and subjected to immunoblot analyses. For these studies EGFR and calnexin were used as markers of the PM and ER, respectively. As shown in Figure 10A, enriched ER and PM fractions were obtained as indicated by a decrease of EGFR in the ER fraction and an increase in EGFR in the PM fraction. However, the greatest enrichment was achieved for IB, which was only detected in the fraction corresponding to the PM marker. These results suggest that IB is preferentially associated with the plasma membrane and not the ER. As shown in Figure 10B, in PM fractions isolated from irradiated U251 cells, there was a significant loss of both IB and p65 NF-B. Thus, these results further suggests that the IR-sensitive pool of IB/NF-B is located in the plasma membrane.

View larger version (27K): [in this window] [in a new window]   Figure 10.   IB and p65 NF-B protein levels in isolated ER and PM fractions of U251 cells. (A) Immunoblot analysis of isolated PM and ER fractions for protein markers (EGFR and calnexin, respectively) and IB. (B) Cells were mock irradiated or irradiated with 10 Gy, collected after 2 h, and the PM fraction isolated. Immunoblot analysis was then performed using actin as a loading control. W, whole cell lysate.

However, because of an inability to more completely reduce the ER component in the PM fraction as shown in Figure 10, confocal microscopy was used as an additional approach to investigate the subcellular location of IR-sensitive IB. For this study cells were grown on chamber slides and immunocytochemistry performed using an EGFR antibody to demarcate the PM (green fluorescence) and an antibody to IB (red fluorescence). As shown in Figure 11, in control untreated U251 cells IB was distributed throughout the cell. Treatment with TNF- resulted in the complete loss of IB, consistent with the immunoblot analysis shown in Figure 2A. In contrast, IR seems to result in the loss of IB primarily around the periphery of the cell. Thus, these data along with the cell fractionation studies suggest that IR selectively targets IB associated with the plasma membrane.

View larger version (123K): [in this window] [in a new window]   Figure 11.   Immunofluorescence localization of IB and EGFR (PM marker) in U251 cells after treatment with IR and TNF-. Cells growing in chamber slides were exposed to IR (10 Gy, 2 h) or TNF- (10 ng/ml, 30 min) and then incubated with antibodies to EGFR (green fluorescence) and IB (red fluorescence). Representative cells are shown as imaged using confocal microscopy.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The ability of diverse stimuli such as cytokines, hypoxia, UV radiation, and ionizing radiation to activate NF-B suggests that this transcription factor is susceptible to activation via a number of signaling pathways (Koong et al., 1994; Li and Karin, 1998; Uehara et al., 1999; Gao et al., 2001). Along these lines, Anderson et al. (1994) illustrated the existence of stimuli-specific signaling pathways by showing that oxidative stress (H₂O₂) triggered NF-B activation in only one of two human T-cell lines, whereas cytokines and phorbol esters stimulated NF-B in both cell lines. Although the specific processes mediating activation by TNF- have been well defined, the signaling events involved in NF-B activation after other stimuli have not been fully elucidated. NF-B activation by hypoxia involves tyrosine, rather than serine phosphorylation and is accompanied by IB degradation (Koong et al., 1994). In contrast, Imbert et al. (1996) reported that treatment of Jurkat T cells with pervanadate resulted in tyrosine phosphorylation of IB and activation of NF-B independently of IB degradation. Finally, UV-C was shown to activate NF-B in the absence of an increase in IKK activity (Li and Karin, 1998). Defining the individual signaling pathways that lead to the activation of NF-B would not only be of fundamental value but may also provide the basis for approaches that inhibit NF-B activation by a specific agent, yet would not affect NF-B activation generated by other stimuli.

With respect to IR, it was initially noted that NF-B activation occurs in the absence of IB degradation, that is, based on the analysis of whole cell lysates. The ability of IR to activate NF-B in the absence of IB degradation suggested that IR may, in fact, activate a signaling pathway similar to that of pervanadate involving the tyrosine phosphorylation of IB (Imbert et al., 1996). However, we recently showed that exposure of U251 cells to IR resulted in the serine phosphorylation of IB (Russell et al., 2002). Furthermore, and more convincingly, data in this report (Figure 4) revealed that proteosome inhibition significantly reduced activation of NF-B after IR. These results indicated that IB degradation was indeed a critical step in IR-induced NF-B activation, but undetectable using standard approaches.

A possible explanation was that IR was affecting a specific, smaller pool of IB. This would be consistent with the relative inefficiency of IR as an activator of NF-B compared with TNF- (Li and Karin, 1998; Shao et al., 2001). Whereas the classical paradigm of NF-B activation defines IB as a cytoplasmic protein (Karin and Ben Neriah, 2000), several reports have shown that IB can localize or interact with subcellular compartments other than the cytoplasm. IB has been detected in the nucleus where it associates with NF-B dimers and returns them to the cytoplasm as inactive complexes (Hay et al., 1999; Rodriguez et al., 1999; Sachdev et al., 2000). In addition, Cuervo et al. (1998) demonstrated that in Chinese hamster ovary cells IB could also be found in lysosomes; under conditions of nutrient deprivation lysosomal IB was degraded resulting in the activation of NF-B. Finally, IB can also exist in the mitochondria associated with ANT, a mitochondrial ATP transport protein (Bottero et al., 2001b). To determine whether IR affected a subcellular pool of IB other than that located in the cytoplasm, U251 cell lysates were first fractionated into insoluble and soluble components. These initial studies clearly showed that IR resulted in a loss of IB in the insoluble cell fraction without having any effect on IB in the soluble portion (i.e., the cytoplasmic pool). This is in contrast to treatment with TNF-, which degraded IB in both fractions. However, as shown in Figure 5, the majority of IB is localized in the soluble cell fraction consistent with previous studies (Karin and Ben Neriah, 2000), which explains the failure to detect IB degradation after IR when using the standard approach of evaluating whole cell lysates. Regarding the other members of the IB protein family, whereas IB was detected in both the soluble and insoluble fractions, IB was only found in the soluble compartment. Furthermore, IB was degraded after exposure to TNF-; IB was not affected by treatment with TNF- or IR. These variations in location and susceptibility to stimuli may reflect the different roles of the IB family members in the regulation of NF-B activity (Baeuerle and Baltimore, 1996; Renard et al., 2000).

The localization of IR-sensitive IB to the insoluble protein fraction was shown for the U251 and SF539 cells lines, which are both susceptible to IR-induced NF-B. Interestingly, in the BXPC-3 cell line, which does not activate NF-B in response to IR, IB was only detected in the soluble and not in the insoluble cell fraction. Although these results were generated from only three cell lines, they suggest that IB localization plays a role in determining the susceptibility to IR-induced NF-B activation. However, it should be noted that in HeLa cells, Li and Karin (1998) showed that IR-induced NF-B activity is accompanied by IB degradation in whole cell extracts, although to a smaller extent than detected after TNF- treatment. This may reflect cell type specificity or different levels of IB in the insoluble fraction, such that their loss is not masked by the "dominant" soluble fraction.

Investigations using serial centrifugation narrowed the locale of IR-sensitive IB to the ER and/or PM fractions. Further studies attempting to obtain enriched ER and PM fractions combined with confocal microscopy after immunocytochemical analyses suggested that the IR-susceptible IB/NF-B complex is located at the plasma membrane. This was unexpected given that previous work seemed to suggest that IB found in the nucleus or mitochondria would be of the most biological significance, i.e., after that found in the cytoplasm (Hay et al., 1999; Rodriguez et al., 1999; Sachdev et al., 2000). However, although clearly detected in these organelles of U251 cells, the IB found in the nuclei and mitochondria, like that in the cytoplasm, was not sensitive to IR-induced degradation. The membrane association of IB is actually consistent with previous results showing that a single chain antibody fragment targeted to Ras protein inhibits IR-induced NF-B activation in U251 cells and other cell lines (Russell et al., 2002). This inhibition only occurred in cells that expressed an active form of Ras (Russell et al., 2002). Thus, a possible scenario is that active Ras proteins are involved in recruiting IB/NF-B complexes to the cell membrane. It should be noted that BXPC-3 cells, which do not activate NF-B after IR and do not have IB in the insoluble fraction, also do not contain significant levels of activated Ras (Russell et al., 2002).

The studies presented herein demonstrate that, whereas TNF- treatment results in degradation of all cellular IB, including the dominant pool in the cytoplasm, IR-induced degradation is selective for IB located in the insoluble cell component, putatively at the PM. One of the critical questions then raised by this study pertains to the resistance of the cytoplasmic pool of IB to IR-induced degradation. A possible explanation may lie in the multiple effects of TNF-. This cytokine interacts with its specific receptors, which have been mechanistically linked to the signaling pathway regulating IKK and subsequent IB phosphorylation and degradation (Pimentel-Muinos and Seed, 1999). However, TNF- treatment also generates reactive oxygen species (ROS) (Chandel et al., 2001; Kohler et al., 2001) with antioxidant treatment inhibiting TNF--induced NF-B activation (Legrand-Poels et al., 1997). In contrast, IR does not serve as a specific receptor ligand, but it does generate ROS. Thus, the situation may exist in which the soluble (cytoplasmic) pool of IB is activated via specific receptor-mediated signaling complexes, but the insoluble pool of IB is activated as a result of ROS generation. Thus, whereas IR fails to activate the receptor-mediated signaling pathway necessary for degradation of the soluble pool of IB, it does generate ROS sufficient for activation mediated by the insoluble pool of IB. In contrast, TNF- activates both pathways and is considerably more efficient at activating NF-B. Clearly, additional studies are needed to define the specific signaling pathways mediating IR-induced NF-B activation. The data presented herein, however, do illustrate that an additional level of regulation of NF-B activation involves subcellular localization.