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Vitamin A status significantly alters nuclear factor-B activity assessed by in vivo imaging

LIV M. I. AUSTENAA^,1, HARALD CARLSEN^,1, AASE ERTESVAG, GEORGE ALEXANDER, HEIDI K. BLOMHOFF and RUNE BLOMHOFF^,2

Department of Nutrition,
Department of Biochemistry, Faculty of Medicine, University of Oslo, Norway

²Correspondence: Department of Nutrition, P.O. Box 1046 Blindern, 0316 Oslo, Norway. E-mail: rune.blomhoff{at}basalmed.uio.no

SPECIFIC AIM

Vitamin A deficiency is characterized by abnormal signaling through the classical pathway (via binding of retinoic acid to the retinoic acid receptor-retinoid X receptor complex specifically associated to a retinoic acid response element in the regulatory region of retinoic acid target genes) as well as other unresolved pathways. The purpose of our study was to investigate, in vivo, the relationship between vitamin A status and the activity of the transcription factor nuclear factor-B (NF-B), a transcription factor central to regulating inflammatory and immune responses, using a novel transgenic murine NF-B-luciferase reporter model that enables molecular imaging of NF-B activity in live mice.

PRINCIPAL FINDINGS

1. Elevated NF-B activity in vitamin A-deficient mice
To study how vitamin A modulates NF-B activity, we measured luciferase activity in NF-B-luciferase reporter mice fed a control or vitamin A-deficient (VAD) diet. Transgenic mice contain the luciferase gene coupled to a promoter with three NF-B-DNA binding sites (B sites) identical to B sites in the immunoglobulin -light chain enhancer region. Hence, luciferase activity reflects NF-B’s trans-activity.

Significantly higher basal level of NF-B activity (basal: without exogenous induction) was found in vitamin A-deficient mice (n=10) compared with control mice (n=11) (Fig. 1 ). When light intensities from whole mice were quantified, vitamin A-deficient mice had 2.2 ± 1.1 (mean±SD) -fold stronger luminescence than did control mice (Fig. 1A ). Analysis of various anatomical regions showed that the luminescence in vitamin A-deficient mice compared with controls was significantly higher in the thoracic region (reflecting the thymus) (2.2±1.0-fold), abdominal region (2.6±1.6-fold), and ventral portion of the paws (Fig. 1A ). Curiously, this increase was particularly manifested in paws from the right side vs. those from the left: light intensities from right front- and hindpaws in vitamin A-deficient mice were 2.9 ± 1.4-fold higher than corresponding paws in control mice, whereas luminescence in paws from the left side increased only 1.7 ± 1.3-fold compared with control mice, suggesting handedness. Luminescence from an area representing the skin was 2.4 ± 1.2-fold higher in vitamin A-deficient mice than in controls. Lymph nodes in the neck region (superficial cervical nodes) readily observed in intact mice tended to have higher luminescence than control mice, but the data were not statistically significant.

View larger version (28K): [in this window] [in a new window]   Figure 1. Elevated luciferase activity in vitamin A-deficient mice relative to controls measured by in vivo imaging. A) Upper panel: luciferase activity in an NF-B-luciferase transgenic control (n=11) and a vitamin A-deficient (n=10) mouse measured by video imaging (VIM). Lower panel: quantification of the VIM collected luciferase activity in control and vitamin A-deficient mice. *P < 0.05, **P < 0.01. B) Luciferase activity in exposed mice (control: n=10, VAD: n=10) and quantification of VIM collected luciferase activity in the superficial cervical, axillary, brachial, mesenteric, and inguinal lymph nodes. The Mann-Whitney test was used for statistical analysis. **P < 0.01

To identify in more detail the light intensities from specific regions in the whole mouse, we took light-intensified images of exposed mice that under anesthesia had the skin layer cut loose from the peritoneum and folded to the sides (Fig. 1B ). The light intensities from various lymph nodes (inguinal, mesenteric, brachial, axillary, and superficial cervical nodes) were quantified, and found to be 1.8 ± 0.6 (mean±SD) -fold higher in vitamin A-deficient mice (n=5) than in control mice (n=4), determined to be statistically significant (P=0.032 using the Mann-Whitney test).

Analysis of luciferase activity in different internal organs showed there was significantly increased luciferase activity in the thymus (1.3±0.3-fold). Other organs studied (kidney, skin, liver, bone marrow, heart, and spleen) showed no significant difference in luciferase activity between vitamin A-deficient and control mice.

Next, we wanted to explore which cells of the vitamin A-deficient lymph nodes exhibited the elevated NF-B activity. We isolated T cells and B cells from lymph nodes (superficial cervical, brachial, axillary, and inguinal) and measured luciferase activity in isolated cells. Luciferase activity was 2.5 ± 2.4 (mean±SD) -fold higher in T cells from lymph nodes of vitamin A-deficient mice than lymph nodes of control mice (Mann-Whitney test: P=0.026). Luciferase activity was not seen to be different in any other cell populations (B cells or total isolated cells) from the lymph nodes.

2. Retinoic acid transiently represses NF-B activity in control and vitamin A-deficient mice
We investigated whether a single high oral dose of all-trans retinoic acid (at-RA) (50 mg/kg in corn oil) would modulate the increased NF-B activity found in the vitamin A-deficient mice. Six hours after at-RA administration, NF-B-dependent luciferase activity of the whole mouse was reduced by 40 ± 14% (mean±SD) (Fig. 2 ). Control images of the mice were taken the day before at-RA was administered. The luciferase activity in mice given the corn oil vehicle was not altered. Twenty-four hours after at-RA administration, luciferase activity of vitamin A-deficient mice returned to the elevated levels seen before at-RA treatment. All-trans-RA administered to mice fed control diet also exhibited a marked but transient reduction (42±16%) of NF-B activity.

View larger version (41K): [in this window] [in a new window]   Figure 2. Transient down-regulation of NF-B activity in vitamin A-deficient and control mice after all-trans retinoic acid administration. All-trans retinoic acid (50 mg/kg), suspended in corn oil, was administered by oral gavage to vitamin A-deficient mice (n=10). After 6 and 24 h, luciferase activity was measured by video imaging. Quantification of luciferase activity in various regions of the mice was performed. The Wilcoxon test was used for statistical analysis of each region at the different time points. Bars representing luciferase activity from the same region with a different letter above it indicates they are significantly different from each other (P<0.05).

3. RAR-pan-antagonist transiently elevates NF-B activity in mice fed control diet
Next we wanted to see whether we could mimic a vitamin A deficient-related increase in NF-B activity by administering a RAR-pan-antagonist to mice on a control diet (n=5 at time 0, n=4 at 7 and 24 h). Administration of the RAR-pan-antagonist AGN 194310 resulted in a transient increase in basal NF-B activity. The activity was found to be enhanced by 46 ± 28% above basal levels 7 h after administration of the antagonist. After 24 h NF-B activity was no longer significantly different from basal levels.

4. Vitamin A status does not affect p65 DNA binding activity
The elevated NF-B activity in vitamin A-deficient mice could be due to increased NF-B-DNA binding. However, the p65 DNA binding activity in nuclear extracts of the thymus of control mice (n=5), vitamin A-deficient mice (n=5), and vitamin A-deficient mice (n=5) that received retinoic acid revealed no differences in p65 DNA binding in any of the groups of mice. These findings suggest that the effect of vitamin A is on NF-B’s trans-activation potential and not DNA binding.

5. UVB-induced NF-B activity is enhanced in vitamin A-deficient mice
UVB is a strong inducer of NF-B activity, and we have shown in earlier experiments that UVB is a strong activator of NF-B activity in the skin. Vitamin A-deficient (n=8) and control mice (n=8) were shaved on their abdomen region and a small area was exposed to 480 J/m² UVB (peak 296 nm). Twenty-four hours later, the mice were imaged for NF-B activity. In vitamin A-deficient mice luminescence from the UVB exposed area was 1.6 ± 0.7-fold (mean±SD) higher than in control mice, determined to be statistically significant (P=0.038 using the Mann-Whitney test).

CONCLUSIONS

Molecular imaging is a rapidly evolving research discipline in biomedicine with the potential of elucidating crucial biological processes at cellular and subcellular levels in intact organisms. Using our novel transgenic NF-B-luciferase reporter model, we were able to investigate the in vivo relationship between vitamin A status and NF-B activity. To our knowledge, this work is the first demonstration of live-mouse monitoring of dynamic changes in gene regulation as the result of dietary manipulation.

Here we show that a physiological deficiency in vitamin A by itself results in increased basal NF-B activity predominantly in lymphoid organs and the skin. To investigate whether the increased NF-B activity in vitamin A-deficient mice was due to an infection or another pathological state, we performed health screening of a selection of the vitamin A-deficient and control mice (The Microbiology Laboratories, Middlesex, UK). Ten vitamin A-deficient and four control mice were tested for a panel of microorganisms and visible abnormalities. The microbiological report showed no difference in pathogen status in the vitamin A-deficient mice compared with controls. Thus, these tests did not reveal any active infection or another pathological state that could explain the increased NF-B activity observed in vitamin A-deficient mice. Nevertheless, we cannot completely rule out the presence of subclinical infection.

We were able to show that the elevated NF-B activity in vitamin A-deficient mice was transiently down-regulated by supplementation with retinoic acid. Furthermore, the RAR-pan-antagonist AGN194310, mimicking a vitamin A-deficient state, caused a transient elevation of NF-B activity. The experimental results using the RAR-pan-antagonist provided every indication that the repressive activity of retinoic acid on NF-B is mediated directly through nuclear receptors RARs and RXRs. The inhibition of NF-B activity by retinoic acid seemed to occur rapidly, transiently, and directly without affecting NF-B’s DNA binding capacity.

Vitamin A deficiency is a major problem in the developing world, affecting >100 million children, and is characterized not only by impaired vision but also by an increase in morbidity and mortality of certain infections due to an impairment of immune responses. Innate as well as adaptive immune responses are affected. A T helper (Th) 1 cell bias and a diminution of antibody responses directed by Th 2 cells are observed. It has been proposed that the Th1 cell bias seen during vitamin A deficiency is mediated by increased expression of the Th1-promoting cytokines IL-12 and IFN-. While both cytokines are negatively regulated by vitamin A, they are induced by NF-B. The importance of NF-B in promoting and upholding a Th1 response has been demonstrated, although some studies have shown them to be dispensable for a Th2 type response whereas retinoic acid enhances T helper type 2 responses. Hence, we postulate that the elevated NF-B activity observed during vitamin A deficiency may be one of the mechanisms facilitating the Th1 cell bias.

In the present study we have established that reduced or absent vitamin A signal during vitamin A-deficiency leads to elevated NF-B activity (Fig. 3 ). This may be one of the underlying mechanisms for the impaired immune responses observed during vitamin A deficiency.

View larger version (45K): [in this window] [in a new window]   Figure 3. Elevated NF-B activity during vitamin A deficiency may be one of the mechanisms underlying the impairment in the inflammatory and immune responses. During normal vitamin A status, retinoic acid binds to the RAR/RXR complex, induces transcription through an RARE element, and inhibits transcription through a B site by inhibiting trans-activity of NF-B. The latter is most likely independent of an RARE. During vitamin A deficiency there is reduced signaling through the RAR/RXR complex leading to decreased RAR-RXR/RARE-directed transcription and increased NF-B/B site-driven transcription, the latter due to lack of the inhibitory activity of retinoic acid on NF-B.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1098fje; doi: 10.1096/fj.03-1098fje

¹ These authors contributed equally.

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