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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 9, 2005 as doi:10.1096/fj.04-3660fje. |
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,1
,¶
* Endocrine Section, Laboratory of Clinical Investigation, NCCAM, NIH, Bethesda, Maryland, USA;
First Department of Pediatrics, UOA, Athens, Greece;
Clinical Neuroendocrinology Branch, NIMH, NIH, Bethesda, Maryland, USA;
Office of Clinical and Regulatory Affairs, NCCAM, NIH, Bethesda, Maryland, USA;
Department of Pathology, LUMC, Maywood, Illinois; and
¶ Reproductive Biology and Medicine Branch, NICHD, NIH, Bethesda, Maryland, USA
1Correspondence: Endocrine Section, Laboratory of Clinical Investigation, National Center for Complementary and Alternative Medicine, NIH, 9 Memorial Drive, Bldg 9, Room 1N105, Bethesda, MD, 20892, USA. E-mail: manolii{at}mail.nih.gov
SPECIFIC AIMS
This study aimed at the identification of potential gene targets of glucocorticoids that affect mitochondrial function in human skeletal myocytes in search of molecular pathways involved in the pathogenesis of glucocorticoid-induced myopathy. Using a mitochondria-focused cDNA microarray, we observed that dexamethasone significantly up-regulated the gene encoding for the catecholamine-metabolizing enzyme, monoamine oxidase-A (MAO-A), and subsequently explored the effects of dexamethasone on MAO mRNA, protein and enzymatic activity to gain insight into the mechanisms and biological significance of this effect.
PRINCIPAL FINDINGS
1. Detection of significant MAO-A up-regulation by mitochondrial microarray screening in dexamethasone-treated human skeletal myocytes
A novel human cDNA microarray containing 501 nuclear genes related to mitochondrial structure and function (hMitChip) was used to evaluate the glucocorticoid-induced transcriptional adaptations that affect mitochondrial function in human skeletal myocytes in vitro. Skeletal myoblasts derived from vastus lateralis muscle biopsy of a 16-year-old healthy male donor were differentiated to multinucleated myocytes and exposed to 10–6 M dexamethasone for 12 h, 24 h, or 7 days. Microarray analysis was employed to compare gene expression of dexamethasone treated vs. nontreated controls. Dexamethasone altered the expression of genes involved in oxidative phosphorylation, lipid biosynthesis, mitochondrial protein synthesis, and apoptosis (data not shown), with the most significant induction observed for MAO-A, the gene encoding for the primary enzyme involved in catecholamine metabolism (Fig. 1
A, B). Quantitative real-time-RT PCR confirmed a 9.5 ± 1.9-fold increase of MAO-A mRNA levels in dexamethasone treated vs. control cells at 12 h, followed by a 11.5 ± 2.7-fold at 24 h and a 13.9 ± 3.4-fold change after 7 days (all P<0.001). No significant change was observed for the MAO-B transcript (Fig. 1C
).
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2. Dexamethasone elicits a dose- and time-dependent stimulation of MAO-A transcription and translation, with minimal effects on MAO-B
MAO transcripts were estimated by quantitative real-time RT-PCR in skeletal myocytes differentiated from primary myoblasts of 3 healthy male donors (16, 25, and 30 years of age). A linear increase in MAO-A mRNA levels was observed in dexamethasone-treated myocytes, from 1.8 ± 0.2-fold at 10–9M dexamethasone (P<0.0001 vs. nontreated controls) to a maximal 12.4 ± 2.6-fold at 10–6 M dexamethasone (P<0.0001) after 24 h. The EC50 occurred at 10–8 M dexamethasone. Administration of 10–6 M hydrocortisone, prednisolone, and betamethasone induced MAO-A expression that was 67.7 ± 3.1%, 86.1 ± 5.0%, and 101 ± 12.4%, respectively, that was stimulated by dexamethasone (all P<0.001 relative to control). Smaller increases in MAO-B mRNA expression were detected, with a peak of 5.4 ± 2.7-fold at 10–5 M dexamethasone after 24 h (P<0.01).
Incubation of differentiated myocytes with 10–6 M dexamethasone for up to 9 days induced MAO-A mRNA expression to a peak of 21.5 ± 2.2-fold change vs. control (P<0.0001) after 5 days, whereas MAO-B transcripts exhibited a 3.9 ± 1.0-fold change on day 9 (P<0.01). Exposure to the transcriptional inhibitor actinomycin-D (1 or 5 µg/mL) abolished the MAO-A mRNA induction. In contrast, treatment with the protein synthesis inhibitor cycloheximide (10 or 20 µg/mL) exerted no significant effect on MAO-A mRNA expression. Changes in MAO-A protein level, evaluated by Western blot analysis, corresponded closely to those of mRNA. A maximal 6.9 ± 1.1-fold induction was observed after 7 days of treatment with 10–6 M dexamethasone (P<0.05). Coexposure to cycloheximide inhibited dexamethasone-induced MAO-A protein expression in a dose-dependent manner.
3. MAO-A transcriptional activation requires a functional glucocorticoid receptor and Sp1 transcription factor
To explore the underlying mechanisms of the observed transcriptional effects of dexamethasone on MAO-A, we examined whether involvement of the glucocorticoid receptor (GR) was required. Concurrent treatment with the GR antagonist RU 486 (10–6 M) decreased dexamethasone-induced MAO-A mRNA expression by 80% (P<0.0001) and abrogated the increase in MAO-A protein levels (P<0.05).
Given the absence of reports on consensus glucocorticoid response elements (GRE’s) in the MAO-A promoter, we explored the possible role of Sp1 transcription factor, a well-described regulatory factor of both MAO-A and MAO-B transcription. Treatment of myocytes with mithramycin, a specific inhibitor of Sp1 binding to GC-rich regions of gene promoters, inhibited dexamethasone-induced MAO-A expression by 45% and 88% at doses of 0.1 µM (P<0.01) and 1 µM (P<0.001) mithramycin, respectively.
4. Dexamethasone stimulates MAO-dependent hydrogen peroxide production in skeletal myocytes, an effect blocked by selective MAO-A inhibitors
Mitochondrial MAO enzymes catalyze the oxidative deamination of biogenic or dietary amines and xenobiotics, with subsequent release of reactive aldehydes and H2O2. Consequently, we evaluated the effect of dexamethasone treatment on MAO-A-dependent H2O2 production. Significantly higher MAO enzymatic activity and H2O2 levels were observed in dexamethasone-treated vs. control myocytes, which reached a peak of 2.3 ± 0.4-fold vs. nontreated controls after 9 days of exposure to 10–6 M dexamethasone (P<0.01). Addition of a MAO-A specific inhibitor, clorgyline, blocked dexamethasone-induced production of H2O2, whereas addition of a MAO-B specific inhibitor, pargyline, elicited no significant effect (Fig. 2
).
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CONCLUSIONS AND SIGNIFICANCE
Skeletal myopathy is a common and debilitating complication of endogenous and exogenous glucocorticoid excess, yet the underlying pathophysiology remains incompletely understood and targeted therapeutic interventions are limited. Mitochondrial dysfunction and oxidative stress appear to play important pathogenetic roles in glucocorticoid-induced skeletal muscle damage, and treatment with antioxidant enzymes or compounds, such as superoxide dismutase, ascorbic acid, or N-acetylcysteine, has been reported to prevent dexamethasone-induced apoptosis.
This study used a customized, human mitochondria-focused gene chip to identify and evaluate possible molecular targets of glucocorticoids in skeletal myocytes. We now report that of 501 mitochondria-related genes in our hMitChip, MAO-A was the most significantly up-regulated gene in human skeletal myocytes after dexamethasone treatment. To our knowledge, the current study is the first report of a dose- and time-dependent stimulation of the MAO-A mRNA and protein expression by dexamethasone in human skeletal muscle cells. MAO-A transcriptional induction was also observed with other glucocorticoids (hydrocortisone, prednisolone, and betamethasone), and at concentrations that occur in human physiology and pharmacology, which further supports MAO-A as a major target of glucocorticoid action in human skeletal muscle cells.
The mitochondrial enzyme MAO catalyzes the oxidative deamination of neuroactive, vasoactive (serotonin, dopamine, catecholamines), or dietary (tyramine) amines and xenobiotics, releasing reactive aldehydes and H2O2. There are two isoforms of the enzyme, MAO-A and MAO-B, that are encoded by different genes on the X-chromosome and are distinguished by their differential affinity for substrates, sensitivity to inhibitors, and tissue distribution. In contrast to the considerable number of studies of MAO in the central nervous system, little is known regarding the physiology and pathophysiology of MAO-A and MAO-B in skeletal muscle. Regulation of MAO by glucocorticoids has been reported in various tissues other than muscle, mostly in animal studies; however, the detailed mechanisms and biological significance of these effects remain unknown.
Both MAO-A and MAO-B use Sp1 as their essential transcription factor, but they exhibit different promoter properties, possibly contributing to their differential tissue expression. We are unaware of any reports documenting the presence of GRE(s) in the enhancer region of either the MAO-A or MAO-B gene promoter. We now demonstrate that MAO-A transcriptional activation requires both functional GR and the Sp1 transcription factor, as blockade of the GR with RU 486 or ablation of Sp1 binding to the DNA with mithramycin abrogate dexamethasone-mediated effects. The latter findings are consistent with the known effects of dexamethasone on the ubiquitine C promoter, which also lacks a GRE, but contains Sp1 sites and is positively regulated by glucocorticoids through a mechanism involving Sp1 and MEK1.
In addition to the transcriptional and translational stimulation of MAO-A by dexamethasone, our study demonstrated increased MAO-A activity, resulting in higher levels of H2O2 production in dexamethasone-treated myocytes, which was blocked by clorgyline, a specific, irreversible MAO-A inhibitor. Increased MAO-A activity is associated with toxicities in various cell types, mediated in part by the two products of the catalytic reaction: H2O2 and/or the reactive deaminated catecholaldehyde metabolites. MAO-B-dependent H2O2 overproduction contributes to the degeneration of nigral cells in Parkinson’s disease and the tissue damage in the proximal renal tubule during renal ischemia/reperfusion, both of which can be prevented by MAO-B inhibitors. Of note, the MAO inhibitor, tranylcypromine, was recently described as the most effective among various antidepressants in protecting against dexamethasone-induced neuronal damage in rat hippocampus and striatum in vivo.
Given the aforementioned reports of involvement of MAOs and H2O2 in tissue damage in several different organs, we suggest that MAO-A expressed in response to glucocorticoid administration plays an important role in glucocorticoid-induced toxicity in skeletal myocytes by producing the downstream effector molecule, H2O2 (Fig. 3
). Further research appears warranted to explore the role of this pathway in apoptosis and cell damage after exposure of skeletal muscle to glucocorticoids, and the potential use of selective MAO-A inhibitors as a novel pharmaceutical approach for the prevention or treatment of glucocorticoid-induced myopathy. The relevance of our findings to conditions in which chronic mild hypercortisolism is often associated with muscle loss, such as chronic stress, melancholic depression, or the metabolic syndrome and aging, remains to be determined.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3660fje;
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) or -B (
) specific inhibitors (clorgyline and pargyline, respectively) confirmed MAO-A as the MAO isoform responsible for increased H2O2 production. Data are presented as mean ± SE fold change of treated vs. untreated myocytes of 3 separate experiments performed in triplicate (**P<0.01).