Regulation of IκBα expression involves both NF-κB and the MAP kinase signaling pathways
© Zhang et al; licensee BioMed Central Ltd. 2005
Received: 24 March 2005
Accepted: 05 October 2005
Published: 05 October 2005
IκBα is an inhibitor of the nuclear transcription factor NF-κB. Binding of IκBα to NF-κB inactivates the transcriptional activity of NF-κB. Expression of IκBα itself is regulated by NF-κB, which provides auto-regulation of this signaling pathway. Here we present a mouse model for monitoring in vivo IκBα expression by imaging I κB α-luc transgenic mice for IκBα promoter driven luciferase activity. We demonstrated a rapid and systemic induction of IκBα expression in the transgenic mice following treatment with LPS. The induction was high in liver, spleen, lung and intestine and lower in the kidney, heart and brain. The luciferase induction in the liver correlated with increased IκBα mRNA level. Pre-treatment with proteasome inhibitor bortezomib dramatically suppressed LPS-induced luciferase activity. The p38 kinase inhibitor SB203580 also showed moderate inhibition of LPS-induced luciferase activity. Analysis of IκBα mRNA in the liver tissue showed a surprising increase of the IκBα mRNA after bortezomib and SB203580 treatments, which could be due to increased IκBα mRNA stability. Our data demonstrate that regulation of IκBα expression involves both the NF-κB and the p38 signaling pathways. The I κB α-luc transgenic mice are useful for analyzing IκBα expression and the NF-κB transcriptional activity in vivo.
IκBα is an inhibitor of nuclear transcription factor NF-κB, which regulates the expression of proinflammatory and cytotoxic genes . In nonstimulated cells NF-κB proteins are present in the cytoplasm in association with specific inhibitors IκBα, IκBβ and IκBγ. Stimulation by extra-cellular inducers results in the phosphorylation and degradation of IκB through a ubiquitin-proteasome pathway, allowing NF-κB to translocate into the nucleus to activate the transcription of target genes [2, 3]. The IκBα gene contains functional NF-κB sites in the promoter region. Transcriptional activation of IκBα expression by NF-κB leads to rapid re-synthesis of IκBα protein and blockade of NF-κB nuclear translocation [4, 5]. This auto-regulatory loop is both sensitive to and rapidly influenced by NF-κB activating stimuli . In addition, phosphorylation of IκB kinase and the activation of NF-κB also involve the MAP kinase signaling pathways .
In this paper we describe and characterize an I κB α-luc transgenic mouse that was used for monitoring IκBα expression through bioluminescent imaging. We tested the effect of bortezomib and several MAP kinase inhibitors on LPS-induced IκBα expression. The results that follow suggest that, in addition to NF-κB, the MAP kinase signaling pathway is involved in controlling IκBα expression.
Materials and methods
Construction of pIκBα-luc vector and generation of I κB α-luc transgenic mice
A mouse BAC clone containing the mouse IκBα gene was isolated from a CT7 mouse BAC library (Invitrogen, Carlsbad, CA). A 11.0 kb promoter fragment containing sequences 5' to the first ATG for the mouse IκBα gene was obtained by the RED cloning method  and cloned upstream of the firefly luciferase gene in the pGL3-Basic vector (Promega, Madison, WI). A 0.8 kb human β-globin intron 2 was placed between the IκBα promoter and the luciferase gene to optimize the luciferase expression in transgenic mice. The transgene cassette was separated from the vector backbone sequences and used for pronuclear injection into Balb/C mouse strain embryos. These steps yielded the transgenic model henceforth designated Balb/C-Tg(I κB α-luc)Xen and abbreviated in the text as I κB α-luc.
We purchased bacterial lipopolysaccharide (LPS, from Salmonella abortus equi), PD098580 from Sigma-Aldrich Chemical Co., (St. Louis, MO), Bortezomib (VALCADE, PS-341) from Millennium Pharmaceuticals, Inc. (Cambridge, MA), SB203580 from EMD Biosciences, Inc. (La Jolla, CA) and SP600125 from A.G. Scientific, Inc. (San Diego, CA).
In vivo imaging of luciferase activity
In vivo imaging was performed using an IVIS® Imaging System 100 Series (Xenogen Corp., Alameda, CA). I κB α-luc transgenic mice were anesthetized with isoflurane and injected intraperitoneally with 150 mg/kg of luciferin (Biosynth, A.G., Switzerland). Ten minutes after the luciferin injection, mice were imaged for 1–10 seconds. Photons emitted from specific regions were quantified using Living Image® software (Xenogen Corp.). In vivo luciferase activity is expressed as photons/second/cm2.
Study of in vivo IκBα gene regulation using I κB α-luc transgenic mice
I κB α-luc transgenic mice of 3–6 months of age were injected with LPS (1 mg/kg, i.p.). Control mice were injected with saline. At selected time points, mice were imaged for the luciferase signal. To test the effect of various compounds, mice were pre-treated with bortezomib (1 mg/kg, i.v.), PD098059 (10 mg/kg, i.v.), SP600125 (20 mg/kg, i.v.), or SB203580 (5 mg/kg, i.v.) 1 hour prior to the LPS injection.
Tissue luciferase activity
Selected organs were removed and homogenized in 3 volumes of PBS containing a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN) and lysed with passive lysis buffer (Promega). After centrifugation at 14,000-rpm for 10 min at 4°C, the supernatant was collected. Luciferase activity was assayed using the Luciferase Assay System (Promega) and a Turner Design, TD 20/20, Luminometer (Sunnyvale, CA). Protein concentration was estimated with Bradford reagent (Sigma-Aldrich).
Northern blot analysis
Total RNA was isolated from mouse tissue using RNAwiz (Ambion, Austin, TX) and further purified using the RNAeasy kit (Qiagen Inc., Valencia, CA). A total of 2 μg of RNA sample was analyzed by Northern blot using a NorthernMax system (Ambion). A 482 nt IκBα cDNA fragment was amplified (forward primer: 5'- GCTCTAGAGCAATCATCCACGAAGAGAAGC-3'; reverse primer: 5'- CGGAATTCGCCCCACATTTCAACAAGAGC-3') and cloned into the pBlueScript SK vector (Stratagene, La Jolla, CA) that was linearized with XbaI and EcoRI. Single strand antisense IκBα RNA probe was prepared by transcription with T7 polymerase using a Strip-EZ kit (Ambion). After hybridization, the signal was detected using a BrightStar BioDetect kit (Ambion)
Nonparametric tests for significance were used to test whether changes in luciferase signal from baseline were significantly greater than zero within groups (sign test) and whether the changes from baseline were significantly different between treatment groups (Mann-Whitney test). Values are presented as means ± one standard error in the graphs and text unless otherwise noted. For some statistical tests genders were combined to increase sample number in each group. All significance levels are two-sided.
Induction of I κB α expression by LPS
Luciferase signals from the abdominal region of LPS-treated mice were quantified using the Living Image® software to produce the data shown in Figure 1B. At the peak of induction 2 to 4 hours after injection, the luciferase signals were increased 6 to 10-fold by LPS as compared with basal luciferase signal at T = 0 hour. At 24 hours, the luciferase signal was still 2 to 3-fold greater than basal levels.
IκBα expression is induced in multiple tissues after LPS treatment
Ex vivo measurement of luciferase activity (Unit/μg protein). Selected organs were harvested from IkBα-luc mice that were untreated (control, n = 3) or treated with LPS (1 mg/kg, i.p., n = 3) at 4 hours prior to the harvesting.
Mean ± SE
157 ± 30
3651 ± 48*§
157 ± 2
2933 ± 69*
363 ± 69
6906 ± 878*
218 ± 58
6203 ± 1414*
430 ± 112
3549 ± 291*
348 ± 52
2718 ± 452*
89 ± 39
4640 ± 601*
73 ± 9
1367 ± 598*
65 ± 7
709 ± 62*
67 ± 5
414 ± 26*
15 ± 2§
951 ± 141*
7 ± 2
405 ± 8*
72 ± 13
513 ± 84*
73 ± 9
384 ± 52*
We further attempted to establish a correlation between luciferase activity and IκBα mRNA expression. In the liver tissue of un-treated mice, IκBα mRNA expression was detectable. Following LPS treatment, an induction of IκBα mRNA expression was observed (Figure 1C), which correlated with the increase of luciferase activity in the liver.
Bortezomib inhibited LPS-induced IκBα expression
Bortezomib inhibited LPS-induced I κB α expression in all the organs except the brain
We further examined the effect of bortezomib on IκBα mRNA induction by LPS. In both male and female mice, pre-treatment with bortezomib increased LPS-induced IκBα mRNA level in the liver tissue (Figure 3C).
Effect of the MAP kinase inhibitors on IκBα induction by LPS
The effect of SB203580 on IκBα mRNA induction by LPS is shown in Figure 5B. Pre-treatment with SB203580 increased LPS-induced IκBα mRNA level in the liver tissue of the I κB α-luc mice.
The mouse IκBα promoter contains 6 putative NF-κB binding sites that mediate the NF-κB regulation . Induction of I κB α-luc expression in the early stage of the LPS response is consistent with a tight auto-regulation of the NF-κB signaling pathway by IκBα . By reflecting NF-κB transcriptional activity, the luciferase signal in the I κB α-luc mouse provides a convenient approach for in vivo monitoring of NF-κB activation.
It has been shown previously that LPS treatment causes degradation of IκBα protein within 40 minutes, followed by induction of IκBα mRNA that results in rapid recovery of the IκBα protein by 3 hours. As a result, maximal NF-κB activation occurred 1 hour after LPS treatment but started to decline at 3–6 hours post treatment . In agreement, our in vivo imaging data demonstrated an induction of luciferase activity at 2 to 4 hours after treating the I κB α-luc mice with LPS, followed by decline of the luciferase activity at 7 and 24 hours. In addition, we also observed a slight gender difference of the kinetics of NF-κB activation following LPS treatment. Male mice showed a peak of induction at 4 hours, followed by a sharp decrease at 7 hours. Female mice showed a peak of induction at 2 hours, followed by a sequential decrease at 7 and 24 hours. This indicates that LPS-induced inflammation process may be sustained longer in female mice than in male mice.
Ex vivo analysis of selected tissues of I κB α-luc mice showed baseline luciferase expression in liver, spleen and lung, with lower expression in intestine, kidney, heart and brain. Significant induction of luciferase expression was observed in all of these organs in both male and female mice after LPS treatment, with higher luciferase activity observed in liver, spleen and intestine as compared to other tissues (Table 1). This is consistent with the bioluminescent imaging analysis of luciferase activity in the live mice that shows higher luciferase signals were present in both hepatic and intestinal regions than other parts of the body (Figure 1A). High extent of luciferase induction in the liver, spleen, lung and intestine by LPS is consistent with IκBα degradation and NFκB activation in these organs in response to endotoxemia [11–13]. When male and female mice are compared, the luciferase signal in intestine was significantly higher in the LPS-treated male mice as compared with the female mice. The difference could be due to the difference of the kinetics of luciferase induction between male and female mice or simply due to a relatively small sample number used for this study.
Bortezomib inhibited LPS-induced luciferase activity by 70–80% in the I κB α-luc mice, which is confirmed by a broad suppression of luciferase activity in all the analyzed tissues except the brain. Bortezomib is an inhibitor of proteasome activity that is required for IκB degradation and subsequent nuclear translocation of NF-κB . In addition, bortezomib can also inhibit other cell signaling pathways, such as mitogen-activated protein kinase growth signaling, causing inhibition of cell proliferation and induction of cell apoptosis [15, 16]. Analysis of the IκBα mRNA showed that bortezomib pre-treatment caused a further increase of LPS-induced IκBα mRNA in the liver. Since the transcriptional activity of the IκBα promoter was suppressed bortezomib, we suspect that the increase of IκBα mRNA after bortezomib treatment should be due to an increase of IκBα mRNA stability. These data suggest that inhibition of NF-κB mediated inflammation by bortezomib may be due to a broad range of effects, affecting processes such as IκB protein degradation and IκBα mRNA stability.
Several MAP kinase inhibitors were tested for their effect on LPS-induced NF-κB activation. We demonstrated that pre-treatment with p38 MAP kinase inhibitor SB203580 at a dose of 5 mg/kg partially inhibited LPS-induced luciferase expression in the I κB α-luc mice in liver, lung and intestine. It has been reported that SB203580 inhibits inflammatory cytokine production in vivo in both mice and rat with IC50 value of 15 to 25 mg/kg . In another report, it was shown that SB203580 at 5, 10 and 20 mg/kg produced a dose dependent inhibition on TNF-alpha production in vivo . Therefore, it is likely that the SB203580 dose used in our study had an inhibitory effect on p38 MAP kinase activation. We also showed that the ERK MAP kinase inhibitor PD098059 at 10 mg/kg partially inhibited LPS-induced luciferase expression at 7 hours. At this dose, PD098059 was able to suppress ERK1/2 phosphorylation in vivo . We further showed that JNK kinase inhibitor SP600125 at 20 mg/kg had no effect on LPS-induced luciferase expression. At this dose, SAPK/JNK MAP kinase phosphorylation can be totally inhibited in the liver tissue .
In summary, we have produced a transgenic mouse in which luciferase expression is driven by the IκBα promoter. We observed a ubiquitous expression and induction of IκBα in the I κB α-luc transgenic mice by LPS. We demonstrated involvement of both the NF-κB and the p38 MAP kinase signaling pathways in the induction of IκBα expression by LPS.
Clinically, NF-κB activation is involved in many chronic disease conditions, such as rheumatoid arthritis, atheroscleorosis, asthma and tumor development [21, 22]. The luciferase activity in the I κB α-luc mice could be used as a sensor for monitoring the NF-κB activation and to further understand how NF-κB activation contributes to the initiation and progression of these disease conditions. In addition, I κB α-luc mice could also be used for testing or even screening of novel NF-κB inhibitors for therapeutic potential.
We thank Paul T. Williams for consulting on the statistical analyses of the data.
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