Nitric Oxide-inducible Expression of Heme Oxygenase-1 in Human Cells

Expression of heme oxygenase-1 (HO-1) in mammalian cells contributes to resistance to various types of free radical damage. Nitric oxide (NO) induces HO-1 in many cell types, but the specific contribution of transcriptional or post-transcriptional effects to this induction have remained unresolved. Here we show that the extent of HO-1 mRNA expression in IMR-90 and HeLa cells depends on the rate of NO delivery, and that the induction occurs more slowly in HeLa than in human fibroblast (IMR-90) cells. We used a specific NO scavenger (2-(4-carboxylphenyl)-4,4,5,5-tetramethylimidazolin-1-oxyl 3-oxide) that completely prevented the inducible expression of HO-1 by NO, pointing to direct signaling action of NO in this induction. By inhibiting transcription during the NO exposure, we have confirmed that NO treatment activates a mechanism that stabilizes HO-1 mRNA. The increase in the HO-1 mRNA half-life in IMR-90 cells was directly correlated with increasing rates of NO release. We also show here that the stabilization of the HO-1 message does not require de novo protein synthesis. Collectively, these results show that stabilization of HO-1 mRNA can be finely tuned to the NO exposure, and that the effect in human fibroblasts is mediated by a pre-existing protein.

In recent years, several groups have investigated the functional significance of HO-1 induction, usually by observing the ability of cells to resist different stress insults when HO-1 is under-or overexpressed. These studies have supported the designation of an important cellular defense role for HO-1 against oxidant injury (13)(14)(15)(16)(17)(18)(19)(20). However, the protective mechanism by which HO-1 acts in different situations is not always clear. One key effect may be the ability of HO-1 to degrade the potentially dangerous intracellular pro-oxidant heme (21). However, HO-1 activity also generates bilirubin as a by-product that can act as a potent peroxyl radical scavenger (22,23). An indirect effect of HO-1 activity could be the induction of ferritin by the iron released by heme degradation; more effective sequestration by ferritin would limit free iron from participation in the Fenton reaction (24,25). Indirect effects could also arise from HO-1-generated CO changing gene expression (26,27). A recent study demonstrated the importance of CO in providing protection against hyperoxic lung injury (28).
The ability of cells to modulate HO-1 expression must certainly contribute to the effectiveness of HO-1 in antioxidant defense. The mechanism of HO-1 induction is therefore of great interest. Most known HO-1 inducers (heme, cadmium, UVA irradiation, hydrogen peroxide, sodium arsenite) seem to increase of the rate of transcription of the HO-1 gene (29 -31). However, some contribution of changes in HO-1 mRNA stability have been seen with nitric oxide (32,33).
NO, which plays roles in cellular signaling and in cytotoxicity, induces HO-1 expression in various cell types by a cGMPindependent pathway (10,(32)(33)(34)(35)(36)(37)(38)(39)(40). Both transcriptional and post-transcriptional mechanisms have been implicated in this induction. One study using sodium nitroprusside, an NO donor, concluded that the induction was principally at the transcriptional level, since no change in the stability of HO-1 mRNA was observed (38). A similar conclusion was drawn by Durante et al. (39), who showed that different classes of NO donors increased HO-1 gene transcription 3-6-fold in vascular smooth muscle cells without a significant change in the half-life of HO-1 mRNA. On the other hand, contribution of both transcription and increased mRNA stability were found for vascular smooth muscle cells treated with an NO-donor compound (32) and for human fibroblasts treated with pure NO gas (33). Thus, changes in HO-1 mRNA stability may contribute to induction of the activity, but the exact mechanism can vary with the cell type and the NO exposure conditions. In this study, we elucidate further the role of mRNA stabilization in the induction of HO-1 by nitric oxide. We have used different NO donors to demonstrate a direct correlation between the NO flux rate and HO-1 mRNA stability in human fibroblasts, and we have determined whether changes in mRNA stability depend on de novo protein synthesis.
Cell Culture-Primary human fibroblasts isolated from embryonic lung, IMR-90 cells, and the HeLa human cervical cancer cell line were generously provided by Dr. Robert Schlegel. These cells were cultured in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclone, Logan, UT). Cultures were maintained at 37°C in a 5% CO 2 humidified atmosphere. All experiments were performed with confluent culture cells. Conditioned medium (CM) was generated by seeding T-162 tissue culture flasks with IMR-90 cells at one-fourth density and growing them to confluence over 3 days. CM was then collected and stored at 4°C until use.
Cell Treatments-Cells were exposed to NONOates with the concentrations and times indicated in the results. To determine the mRNA stability of HO-1 in NO-treated or untreated cells, the inhibitor of transcription AD was added to the cell culture medium at the final concentration of 10 g/ml after the NO treatment. Total RNA was extracted after the times indicated in individual figures. Results obtained with AD were checked with DRB at a concentration of 10 g/ml. When AD was used to study the effect of de novo RNA synthesis on NO-induced HO-1 mRNA stability, it was added at the same time as SPER/NO. However, because the NO treatment time (1 h) was shorter than the transcription inhibition treatment, the medium was changed after 1 h to remove the NO donor, and replaced by CM containing the appropriate transcription inhibitor for further incubation. To study the requirement of de novo protein synthesis on the HO-1 mRNA turnover, two inhibitors of translation were used: cycloheximide (a translation elongation inhibitor) and puromycin (a translation initiation inhibitor). The inhibitor was added to the medium at a final concentration of 10 g/ml, at time 0 in the presence of AD, and total RNA was extracted at time 0, 2, 4, and 6 h. To study the requirement of translation for the stabilization of HO-1 mRNA in response to NO, puromycin and AD were added at the same time as SPER/NO. As mentioned before, media were changed after 1 h to remove SPER/NO and replaced by CM containing the appropriate translation and transcription inhibitors. As DETA/NO and SPER/NO can release by-products other than NO, their possible effect on HO-1 mRNA expression were evaluated by "reverse-order addition" (ROA) experiments. ROA consisted of adding the NO donor to CM to allow it to decompose for 144 h for 1 mM DETA/NO or 48 h for 0.5 mM SPER/NO. Cells were then incubated with these decomposed NO donors as indicated under "Results." NO Measurement-Cells were exposed to either DETA/NO or SPER/NO at concentrations and times indicated, and the rate of NO release was measured in the culture medium by following the disappearance of the DETA/NO and SPER/NO at 251 and 250 nm, respectively. These NONOate compounds (X[N(O)NO] Ϫ ), which emerge from the precise mixture of a nucleophile molecule (X Ϫ ) and NO, spontaneously release 2 mol of NO/mol of dissociating ion (41). In our experiments, these molecules were quantified spectrophotometrically using the extinction coefficients ⑀ DETA/NO ϭ 7,680 M Ϫ1 cm Ϫ1 and ⑀ SPER/NO ϭ 8,000 M Ϫ1 cm Ϫ1 .
Northern Blot Analysis-IMR-90 or HeLa cells were grown in T-25 tissue culture flasks and exposed to NO and other products as described above. After the incubation time indicated under "Results," total RNA was extracted from the cells using a commercially available kit (RNeasy; Qiagen, Valencia, CA). RNA concentrations were measured spectrophotometrically at 260 nm. For each sample, 5 g of total RNA was electrophoresed at 65 mV for 2-3 h in a 1% agarose/formaldehyde gel, transferred to a positively charged nylon membrane, and probed with the 1-kilobase EcoRI fragment of the human HO-1 cDNA (provided by Dr. Rex Tyrrell, University of Bath, Bath, United Kingdom; see also Ref. 5), which was labeled by the random hexamer priming method (Life Technologies, Inc.). After stripping, the blots were then re-probed with a 1.3-kilobase PstI fragment of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA as a loading control. After two washes at room temperature with 2ϫ standard saline phosphate/EDTA (0.15 M NaCl, 10 mM phosphate, pH 7.4, 1 mM EDTA), 0.1% SDS for 15 min and a last washing at 60°C with 0.1ϫ standard saline phosphate/EDTA (0.15 M NaCl, 10 mM phosphate, pH 7.4, 1 mM EDTA), 0.1% SDS for 30 min, the blots were autoradiographed and the intensity of the hybridization signals was quantified with an Instant Imager analyzer (Packard).
Measurement of LDH Release-Cellular toxicity was determined by measuring the release of LDH activity into the culture medium (kit LD-L 10, Sigma Diagnostics). The percentage of LDH released was defined as the ratio of LDH activity in the supernatant to the sum of LDH activities found in the supernatant and in an extract of the cell pellet.

Differential HO-1 mRNA Expression in HeLa and IMR-90 Cells Exposed to Different Fluxes of Released NO-Different
structural families of NO donors have been used to examine the biological function of this gaseous molecule. In this study, members of the diazeniumdiolate family (called NONOates) were used as sources of bioactive NO because of their predictable and various rates of NO release at physiological pH (41). In fact, under our experimental conditions (37°C in DMEM, 5% CO 2 atmosphere), DETA/NO had a half-life of 15 h (data not shown) and therefore slowly generated NO in the culture medium. IMR-90 fibroblasts and HeLa cells were exposed to increasing concentrations of DETA/NO for 8 h to provide rates of NO release ranging from 1 to 30 nM/s (Fig. 1B). HO-1 mRNA expression was then observed by Northern blotting (Fig. 1A). IMR-90 cells expressed a significant basal level of HO-1 mRNA, while HeLa cells did not contain sufficient HO-1 mRNA to detect by Northern blotting in our hands. However, the level of HO-1 mRNA was increased in both cell types after exposure to DETA/NO. Induced HO-1 mRNA was apparent in HeLa cells exposed to 12.5 nM NO/s and in IMR-90 cells exposed to NO at  A). B, the rate of NO release. The decay of the DETA/NO donor was measured spectrophotometrically (as described under "Experimental Procedures") and converted to NO release in nM/s. C, quantitative comparison of NO release and HO-1 mRNA induction. The intensity of the hybridization signals shown in A was quantified, and the HO-1 signals were normalized to those for GAPDH mRNA. The experiments were performed twice (HeLa cells) or three times (IMR-90 cells), and a representative result is shown. 5 nM NO/s (Fig. 1, A and B). The increased expression of HO-1 mRNA, normalized to GAPDH, was directly correlated in both cell types with the rate of NO release (Fig. 1, B and C). The maximal induction in this experiment was seen with 1 mM DETA/NO, which produced NO at 20 -30 nM/s in contact with cells (Fig. 1B).
DETA/NO at 1 mM was used to compare the time course of HO-1 mRNA induction in both cell types (Fig. 2). Under these conditions, DETA/NO released NO at a linear rate equivalent to 24 nM/s (data not shown; cf. Fig. 1B). The results showed that induction of HO-1 mRNA in IMR-90 cells may have occurred somewhat more rapidly, and did achieve a higher maximum level than found for HeLa cells (Fig. 2). Indeed, increased HO-1 mRNA was detected in IMR-90 cells as early as 1 h after starting the DETA/NO exposure whereas ϳ2 h of exposure was required for HeLa cells (Fig. 2). For both cell types, the maximal HO-1 induction occurred at ϳ4 h of exposure (Fig. 2B).
Because NO donors can generate by-products other than NO, we have also tested whether such by-products could affect HO-1 mRNA expression. When 1 mM DETA/NO was completely decomposed in CM for several days and the cells were exposed to this material for 8 h (ROA experiment described under "Experimental Procedures"), there was no change in the level of HO-1 mRNA (Fig. 2, right panel, far right lane). Thus, at least the stable by-products of DETA/NO decomposition are not responsible for HO-1 mRNA induction.
HO-1 mRNA expression was also measured after SPER/NO exposure, which can mimic a burst release of NO. This compound had a half-life of 36 min under our experimental conditions, with 0.5 mM SPER/NO releasing NO at the rate of nearly 120 nM/s (data not shown). HeLa and IMR-90 cells were exposed to SPER/NO for increasing times ranging from 15 to 60 min and analyzed for HO-1 expression. As shown in Fig. 3, HO-1 mRNA expression was not induced in SPER/NO-treated HeLa cells, while it rapidly increased up to 5.5-fold during a 1-h exposure of IMR-90 cells. In an ROA experiment, the effect of 0.5 mM SPER/NO was reduced 8-fold (data not shown), again indicating that stable reaction by-products of NO are not the major inducers of HO-1 mRNA under our conditions. Requirement of Pure NO to Induce HO-1 mRNA Expression-To determine the involvement of NO itself in the induction of HO-1 mRNA expression, we used carboxy-PTIO. Unlike most NO scavengers, which do not discriminate nitric oxide from NO-derived by-products, carboxy-PTIO immediately reacts with NO ⅐ to form the NO 2 ⅐ radical, the closest oxygen-dependent product of NO (41). Thus, by using carboxy-PTIO as a dual-function NO scavenger and NO 2 ⅐ donor, we could test both the requirement for NO⅐ and the possible effect of NO 2 ⅐ and NO 2 ⅐ -derived products, such as N 2 O 4 , NO 2 Ϫ , and NO 3 Ϫ , in induction of HO-1 (42,43). IMR-90 cells were treated with DETA/NO (1 mM, 8 h) in the absence or presence of carboxy-PTIO, then analyzed for HO-1 mRNA. Under our experimental conditions, carboxy-PTIO alone or in combination with DETA/NO did not affect the amount of total RNA recovered (data not shown) or the amount of the GAPDH mRNA present (Fig. 4). Further, only a small increase in the release of LDH was observed under these experimental conditions: control, DETA/NO alone, and carboxy-PTIO alone, all ϳ4% release, versus carboxy-PTIO and DETA/NO combined, 14% release (data not shown). Although carboxy-PTIO itself had a weak but measurable effect on HO-1 mRNA induction (Fig. 4, lane 2), this compound completely blocked the action of DETA/NO (Fig. 4, compare lane 2 versus  lane 4 and lane 1 versus lane 3). We conclude that NO 2 ⅐ and NO 2 ⅐ -derived species are not involved in HO-1 induction in IMR-90 cells, and the data point toward a direct role for NO itself.
Effect of Increasing Rates of NO Release on HO-1 mRNA Turnover-By using NONOates, three different rates of NO release were tested for their effect on the turnover of HO-1 mRNA in IMR-90 cells. We used 0.25 mM DETA/NO to generate NO at 4 nM NO/s, with exposure for 16 h as a "chronic" exposure. A more intense chronic regime was achieved with 1 mM DETA/NO (releasing NO at 30 nM/s) for 8 h. Finally, we used 0.5 mM SPER/NO to generate NO at 120 nM NO/s for 1 h in order to mimic a burst exposure. To determine the mRNA half-life of HO-1, the transcriptional inhibitor actinomycin D was added to the culture medium immediately after NO treatment, and the incubation continued. Untreated-IMR-90 cells

FIG. 3. Time course of HO-1 mRNA induction in HeLa and IMR-90 cells in response to SPER/NO.
A, cells were exposed for the indicated time to 0.5 mM SPER/NO, which generated NO at 120 nM/s under our conditions. Northern blot analysis was performed as described for Fig. 1. B, the level of HO-1 transcript normalized to GAPDH. The results show the mean Ϯ standard deviation for three independent experiments. had a half-life for HO-1 mRNA of 2.3 h (Fig. 5D), similar to that determined previously (33). When the cells were exposed to a chronic exposure of NO at a rate of 4 nM NO/s, the level of HO-1 mRNA was increased and the HO-1 mRNA half-life was not increased significantly (t1 ⁄2 ϭ 2.6 h; Fig. 5A). Exposure to NO at 30 nM/s for 8 h dramatically increased the mRNA level and increased the stability of HO-1 mRNA to a half-life of 6.1 h (Fig.  5B). The stability was further increased to 10.5 h in cells exposed to NO at 120 nM/s (Fig. 5C). The results are summarized in Fig. 5D, which shows the strong correlation between the rate of NO release and the half-life of HO-1 mRNA. We were unable to determine whether NO exposure stabilizes HO-1 mRNA in HeLa cells, because the addition of AD was toxic after 4 h of treatment (data not shown).
Contribution of the Post-transcriptional Events in NO-induced HO-1 mRNA Expression-In order to explore the contribution of post-transcriptional events in the induction of HO-1 mRNA by NO, we blocked transcription by adding AD at the same time as SPER/NO. This approach allowed us to determine whether transcription is necessary for NO-induced stabilization of the HO-1 message. As shown in Fig. 6, the constitutively expressed HO-1 mRNA in IMR-90 cells was rapidly stabilized in response to a burst of NO, and this stability persisted after SPER/NO was removed to give a half-life for the mRNA of 6.3 h. Thus, the mechanism by which NO increases the stability of HO-1 mRNA does not require de novo RNA synthesis.
Effect of Translation on HO-1 mRNA Turnover in Untreated or NO-treated IMR-90 Cells-In order to explore the role of translation in NO-mediated stabilization of the HO-1 mRNA, we employed two inhibitors. We found that the elongation inhibitor cycloheximide by itself increased the stability of the HO-1 mRNA quite strongly (Fig. 7A). Therefore, this agent could not be used to study the role of translation in NO-induced stabilization. However, puromycin, an inhibitor of translation initiation, had little effect on HO-1 mRNA turnover. The halflife of HO-1 mRNA in puromycin-treated cells (2.8 h) was comparable to that in control cells (Fig. 7B). It thus became feasible to study the possible requirement of de novo protein which generated NO at 4, 30, and 120 nM/s, respectively. The medium was then removed, and fresh medium containing 10 g/ml AD was added to the cells. The incubation was then continued. Total RNA was extracted and analyzed for HO-1 and GAPDH mRNA by Northern blotting. After quantitation, the estimated mRNA half-lives were compared for the different conditions (panel D). The data shown are mean and standard deviation of 40 determinations (control), or four determinations (NO generated at 30 or 120 nM/s); for NO generated at 4 nM/s, results for two experiments were averaged and the standard error is shown.

FIG. 6. Effect of the transcription inhibitor actinomycin D on HO-1 mRNA stability induced by SPER/NO.
A, IMR-90 cells were treated with 10 g/ml AD alone, or in combination with 0.5 mM SPER/ NO. After 1 h, the medium containing AD plus SPER/NO was removed and replaced by fresh medium containing AD alone. The indicated times correspond to the total AD incubation. After each incubation, total RNA was extracted and analyzed for HO-1 mRNA expression by Northern blotting. GAPDH mRNA hybridization is shown as a normalization control. B, estimated half-life of HO-1 mRNA. The intensity of hybridization signals in panel A was quantified and is shown. The dotted lines show the estimated half-life values for HO-1 mRNA. The experiment was performed three times, and a representative result is shown.

FIG. 4. Effect of the NO scavenger carboxy-PTIO on HO-1 mRNA induction by DETA/NO.
A, IMR-90 cells were exposed for 8 h to CM, 1 mM carboxy-PTIO, 1 mM DETA/NO alone, or both agents in combination. Under these experimental conditions, the NO donor DETA/NO generated an NO flux of 30 nM/s. Total RNA was then extracted and analyzed for HO-1 and GAPDH mRNA expression by Northern blotting (see Fig. 1). These experiments were performed twice, and a representative experiment is shown. B, the level of HO-1 mRNA normalized to GAPDH; results for two experiments were averaged and the standard error is shown. synthesis during the NO-induced HO-1 mRNA stabilization. We exposed IMR-90 cells simultaneously to 10 g/ml puromycin and AD, in the presence or absence of SPER/NO, and measured the HO-1 mRNA levels using Northern blotting. Our results (Fig. 8) showed that AD-treated cells and AD-puromycin-treated cells presented a similar half-life of the HO-1 mRNA, close to 2 h (Fig. 8, filled circles and filled squares). Most importantly, the HO-1 mRNA stability induced by SPER/NO was still observed when translation was inhibited (Fig. 8, compare open circles and open squares), with the HO-1 mRNA half-life for both AD-SPER/NO-treated cells and ADpuromycin-SPER/NO-treated cells ϳ6 h (Fig. 8). Thus, de novo protein synthesis is not required for NO-induced stabilization of the HO-1 mRNA. DISCUSSION To study the effect of nitric oxide on HO-1 gene expression, various NO-releasing compounds (40) as well as pure NO gas (33) have been used. In the present study, we used two diazeniumdiolates as NO donors because of their controlled and predictable ability to release NO under physiological conditions (44). This approach allowed us to demonstrate a direct correlation between the rate of NO release in the range 1-30 nM/s and the induction of HO-1 mRNA in IMR-90 cells. However, Takahashi et al. (38), using three different classes of NO donors, found a poor correlation between the formation of nitrite (as a measure of NO release) and the HO-1 mRNA level in HeLa cells. They reported that, although sodium nitroprusside released the lowest amount of nitrite (ϳ10 M) compared with the compounds S-nitroso-glutathione (ϳ 15 M) and 3-morpholinosydnonimine (ϳ 40 M), sodium nitroprusside was the most efficient inducer of HO-1 mRNA (38). They obtained similar results in a second study (37). This absence of correlation between the production of nitrite and the induction of HO-1 expression illustrates some problems with use of NO donors, and might be explained as follows. Sodium nitroprusside releases cyanide and iron that may act in additional ways to induce HO-1 mRNA, thus exaggerating the actual NO effect. On the other hand, as 3-morpholinosydnonimine releases NO and superoxide anion simultaneously, the weak effect of 3-morpholinosydnonimine on induction of HO-1 mRNA expression may be due to peroxynitrite formation instead of direct NO production; that would also account for the elevated nitrite release by 3-morpholinosydnonimine. The result from that group may thus be consistent with the data we present here, which point to a role for NO rather than oxygen-dependent NO by-products in HO-1 induction.
Under aerobic conditions, the rapid decomposition of NO can follow different pathways. By interacting with oxygen, NO can successively form the radical NO 2 ⅐ , N 2 O 3 /N 2 O 4 , and then a mixture of nitrite and nitrate. We tested the effect of most of these NO by-products on the induction of HO-1 mRNA by using carboxy-PTIO, which both scavenges NO ⅐ and generates NO 2 ⅐ and its derived species (N 2 O 4 , NO 2 Ϫ , and NO 3 Ϫ ) (43). The results show that NO 2 ⅐ and its related products are not involved in the induction. Although the data are thus consistent with a key signaling role for NO, products such as N 2 O 3 and nitrosothiols cannot be ruled out.
In IMR-90 cells, HO-1 mRNA has a relatively rapid turnover, with a half-life of ϳ2 h. We tested whether active translation was required for HO-1 degradation by using cycloheximide and puromycin, two translation inhibitors with different mechanisms of action. Unexpectedly, HO-1 mRNA was strongly stabilized in cycloheximide-treated cells (half-life Ͼ8 h; Fig. 7A). This result could suggest the involvement of the HO-1 nascent peptide in mRNA degradation, as has been reported for the N-terminal Met-Arg-Glu-Ile motif of ␤-tubulin mRNA in its turnover (45). However, the HO-1 N terminus (Met-Glu-Arg-Pro) differs significantly from that of ␤-tubulin. It was also possible that a labile protein interacts with a cis-regulatory element in the HO-1 mRNA to destabilize it. However, the translation inhibitor puromycin did not significantly stabilize HO-1 mRNA; thus the rapid turnover of HO-1 mRNA does not seem to involve active translation. Since cycloheximide is an inhibitor of translation elongation, it may permit polysome aggregation to stabilize the HO-1 message; in contrast, puro- A, IMR-90 cells were exposed to 10 g/ml AD alone, 10 g/ml AD plus 10 g/ml puromycin, or 10 g/ml AD plus cycloheximide (CHX) for 0, 2, 4, and 6 h. After each incubation, total RNA was extracted and analyzed for HO-1 mRNA expression by Northern blotting. GAPDH mRNA hybridization is shown as a normalization control. B, estimated half-life of HO-1 mRNA. The intensity of hybridization signals in A was quantified and plotted, and the dotted lines show the estimated half-life values. The experiment was performed twice (CHX) or four times (puromycin), and a representative result is shown. mycin dissociates polysomes by causing abortive termination (46). Most importantly, the stabilization of the HO-1 message that is mediated by NO ⅐ does not require translation, as it still occurs in puromycin-treated cells.
The previous result (33) showing stabilization of HO-1 mRNA by pure NO gas led us to study the effects of more physiological doses and long term NO exposure. This approach also avoided the hypoxia that might accompany prolonged (Ͼ2 h) exposures to NO gas (33). Hypoxia also induces HO-1 mRNA expression (7). By using the slow NO-releasing compound DETA/NO and the fast NO-releasing compound SPER/NO, we found a gradual increase in the HO-1 mRNA half-life up to 10.5 h correlated with an increasing rate of NO release up to 120 nM/s. Moreover, the change in message stability occurs immediately upon NO exposure and in the absence of active transcription (Fig. 6). These observations suggest that cells are able to sense and respond directly to various levels of NO by adjusting the stability of HO-1 mRNA. Fine-tuning mRNA stability for HO-1 and perhaps for other transcripts may constitute an important cellular response to NO toxicity. NOinducible stabilization of the HO-1 message is transient, however, and disappears relatively quickly after the NO exposure ceases (33). The failure of others to detect this effect (32,38) may be a result of taking time points too late or using NO levels too low.
The modulation of HO-1 mRNA stability described here is a rapid process independent of RNA or protein synthesis. Thus, a stable protein already present in the cell may modulate HO-1 mRNA turnover in response to NO. One candidate is the iron regulatory protein-1 (IRP-1), which regulates iron metabolism at a post-transcriptional level in mammalian cells (47). Once activated by NO, IRP-1 recognizes specific sequences called iron-response elements (IREs) on several mRNAs (e.g. those encoding ferritin, and the transferrin receptor). In the case of transferrin receptor mRNA, IRE/IRP-1 interaction in the 3Ј end of the message confers stability against endonucleolytic cleavage (48 -49). However, we have not detected IRP-1 binding to any region of HO-1 mRNA in either control or NOtreated IMR-90 cells (data not shown). Further efforts are directed at identifying the relevant control regions of the HO-1 mRNA and the NO-regulated proteins that interact with them.