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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 17, 2003 as doi:10.1096/fj.02-1024fje. |
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,2
* Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA; and
Sezione di Endocrinologia, Dipartimento Clinico Sperimentale di Medicina e Farmacologia, University of Messina, Messina, 98100, Italy
2Correspondence: PREB/NICHD/NIH, 10 Center Dr., Bldg. 10, Room 9D42, Bethesda, MD 20892-1583, USA. E-mail: alescis{at}mail.nih.gov
SPECIFIC AIMS
Evidence from animal and human studies suggests that pharmacologic doses of L-Carnitine (LCAR), a nutrient with a major role in energy metabolism, may mimic some of the biologic actions of glucocorticoids (GLUCs), including their well-known immunosuppressive properties. We hypothesized that this effect could be mediated through activation of the glucocorticoid receptor 
(GR) by LCAR; therefore, we investigated the influence of LCAR on the various properties of the GR, including binding capacity and affinity, intracellular trafficking, and transcriptional and biologic activities.
PRINCIPAL FINDINGS
1. LCAR reduces the binding ability of GR
Whole cell binding (WCB) experiments were performed in HeLa cells. For competitive binding, cells were incubated for 1 h with 25 nM of 3H-dexamethasone (3H-DEX) in the absence (total WCB) and presence of a 500-fold higher concentration of cold DEX (nonspecific WCB) or increasing concentrations of LCAR. For saturation binding experiments, cells were incubated with increasing concentrations of 3H-DEX in the absence and presence of 500-fold higher concentrations of cold DEX or increasing concentrations of LCAR. Specific whole cell binding (SWCB) was calculated by the difference between total WCB and nonspecific WCB.
LCAR suppressed the SWCB of GR to 3H-DEX by 6.5 ± 1.5% at 10 mM (P=0.034), 18.1 ± 4.1% at 25 mM (P=0.004), 25.4 ± 2.0% at 50 mM (P<0.001), and 38.0 ± 2.0% at 100 mM (P<0.001). Saturation binding experiments confirmed the dose-dependent inhibition at different 3H-DEX concentrations. The reduced binding of GR to 3H-DEX in the presence of LCAR was accompanied by a significant dose-dependent increase in the Kd of GR for DEX, with no change in the Bmax.
2. LCAR induces nuclear translocation of GR
The ability of LCAR to influence cell trafficking of GR was tested in HeLa cells transiently transfected with the pF25hGR vector, expressing green fluorescent protein (GFP) -fused human GR. In the absence of LCAR or DEX, GFP-GR was primarily located in the cytoplasm. Addition of either 1 µM of DEX or 50 mM of LCAR to the cell medium triggered translocation of the chimeric receptor from the cytosol to the nucleus within 15 min. After 30 min, GFP-GR was located entirely in the nucleus. This effect of LCAR was dose dependent, since LCAR doses of 25 and 10 mM produced slower translocation of GFP-GR, which was completed in 120 and 240 min, respectively.
3. LCAR stimulates the transcriptional activity of GR
In HeLa cells transiently transfected with the pMMTV-luc vector, which expresses luciferase under the control of the mouse mammary tumor virus (MMTV) promoter, containing four glucocorticoid-responsive elements (GREs), millimolar concentrations of LCAR caused a significant, dose-dependent trans-activation of the viral promoter (Fig. 1
A). Transcriptional stimulation by LCAR was also observed in HeLa cells transfected with the reporter construct TAT3-luc, generated by inserting three synthetic GREs from the tyrosine aminotransferase gene into the pODLO-2 vector, but not in cells transfected with GREs-devoid pODLO-2 (Fig. 1B
). In CV-1 cells, which do not contain functional GR, LCAR was able to trans-activate the MMTV promoter only after exogenous human GR was expressed by cotransfection (Fig. 1C
). Consistent with these results, 1 µM of the competitive GR antagonist RU 486 abolished the stimulatory effect of 100 mM LCAR, as it did with DEX, on the MMTV promoter in HeLa and CV-1 cells.
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4. LCAR suppresses TNF- and IL-12 release from human monocytes in GLUC-like fashion
To evaluate the biologic relevance of our findings, after excluding in vitro cytotoxicity by the transcriptionally active high doses of LCAR, we tested the ability of this compound to mimic the known suppressive effect of DEX on the release of TNF- and IL-12 by human monocytes primed with IFN-
and/or stimulated with LPS ex vivo. Compared with baseline values, 10 nM DEX and 50 and 100 mM LCAR decreased TNF- secretion by 78.8 ± 5.6% (P=0.002), 59.7 ± 7.0% (P=0.027), and 73.0 ± 4.1% (P=0.002), respectively (Fig. 2
A). IL-12 release was suppressed by 67.0 ± 6.0% (P=0.003), 40.3 ± 5.4% (P=0.023), and 44.0 ± 5.4% (P=0.009) of baseline, respectively (Fig. 2B
). The suppressive effect of LCAR and DEX was neutralized by 1 µM of RU 486 (Fig. 2A, B
).
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CONCLUSIONS AND SIGNIFICANCE
LCAR is one of a few compounds that are sold in the United States both as prescription drug and as an over-the-counter nutritional supplement. Currently, the only indication for the use of pharmacologic doses of LCAR (up to 600 mg/kg body weight/day) is the treatment of primary and secondary LCAR deficiencies. However, several potentially beneficial effects of LCAR supplementation have been reported, including modulation of the immune system and other GLUC-like effects.
In the present study, we demonstrated that, at high concentrations, LCAR can trans-activate GLUC-responsive promoters in vitro, similar to DEX. This transcriptional effect of LCAR was dependent on the presence of GREs on the promoter and on the expression of functional GR by the cell, suggesting a common signal transduction pathway for LCAR and DEX. The stimulation of promoter transcription induced by LCAR was suppressed by RU 486, a known competitive inhibitor of GLUCs. Moreover, transcriptionally active doses of LCAR exerted competitive inhibition on DEX binding to GR in HeLa cells by significantly decreasing the affinity of GR for its steroid ligand. Taken together, our results suggest that LCAR may function as an allosteric regulator of GR. The decreased affinity of GR for DEX in the presence of LCAR might be explained by the ability of this nutrient to interact with a portion of the receptor outside the GLUC binding pocket, modifying its allosteric structure. This structural modification would at the same time reduce the affinity of the binding pocket for DEX and create conformational changes similar to those induced by the native ligand, ultimately resulting in GR activation (Fig. 3
). Our hypothesis is also supported by the ability of LCAR to trigger nuclear translocation of the steroid receptor in the absence of GLUCs. However, we cannot exclude the possibility of an indirect effect of LCAR on GR activation through other mechanisms that have yet to be elucidated. The ability of LCAR to reduce GR affinity for DEX, combined with its weaker trans-activating effect compared with DEX, raises the possibility that this compound may act as a partial GLUC agonist/antagonist, able to both trans-activate GR in the absence of the native ligand and antagonize GR activation in its presence.
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Like other anti-inflammatory drugs, GLUCs inhibit the expression of many proinflammatory cytokines. This action is mediated by GR and counteracted by the GR antagonist RU 486. Secretion of the "initial phase" cytokine TNF- and the "immunomodulatory" cytokine IL-12 is consistently reduced by GLUCs, both in vitro and ex vivo. We demonstrated that LCAR, at concentrations that had maximally stimulated the transcription of GLUC-responsive promoters, suppressed the ex vivo release of TNF- and IL-12 by IFN--primed and/or LPS-stimulated human primary monocytes, emulating DEX. The cytokine suppression was abrogated by RU 486, indicating GR dependence of the phenomenon. These results agree with previous reports of immunomodulatory properties of LCAR. However, for the first time, we give a molecular explanation of this effect.
In summary, we provide novel evidence that pharmacologic doses of LCAR, a popular widely available nutritional supplement, can activate GR and modulate the transcription of GLUC-responsive genes in vitro, potentially sharing and/or influencing some of the biologic and pharmacologic actions of these hormones. It was recently reported that, at high concentrations, LCAR had a positive effect on the bone. Indeed, this invites the speculation that pharmacologic doses of LCAR might share the beneficial immunomodulatory properties of GLUCs but not their deleterious effects on the bone. More generally, the modulatory effects of LCAR on GR functions may be tissue and/or gene specific, being influenced by receptor abundance and distribution and/or by transcription regulatory or coregulatory molecules, such as transcription factors, coactivators and/or corepressors. The clinical and therapeutic implications of these findings should be evaluated in controlled trials.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1024fje; doi: 10.1096/fj.02-1024fje 
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