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Glucocorticoid treatment alleviates dystrophic myofiber pathology by activation of the calcineurin/NF-AT pathway

SIMON J. G. ST.-PIERRE^*,, JOE V. CHAKKALAKAL, STEVEN M. KOLODZIEJCZYK^*, JENNIFER C. KNUDSON^*,, BERNARD J. JASMIN^*, and LYNN A. MEGENEY^*,,1

^* Ottawa Health Research Institute, Molecular Medicine Program, Ottawa Hospital, General Campus, Ottawa; and
Department of Cellular and Molecular Medicine and Centre for Neuromuscular Disease, Faculty of Medicine, University of Ottawa, Ottawa Ontario, Canada

¹Correspondence: Ottawa Health Research Institute, Ottawa Hospital, General Campus, Ottawa, ON, Canada K1H 8L6. E-mail: lmegeney{at}ohri.ca

SPECIFIC AIMS

Our aim was to identify the mechanism by which glucocorticoids (such as deflazacort) limit the progression of muscular dystrophy. We examined the JNK1 signaling pathway as a probable deflazacort target based on the observation that elevated activity of this pathway was a contributing factor to dystrophic muscle pathology.

PRINCIPAL FINDINGS

1. Deflazacort attenuates JNK1-induced myocyte loss in vitro by activating the phosphatase calcineurin
In earlier studies we demonstrated that JNK1 is constitutively activated in dystrophic skeletal muscle and that limiting the activity of this kinase in vivo limits the disease pathology. Similarly, skeletal muscle myoblasts that overexpress the JNK1-activating kinase MKK7 recapitulate a loss in myocyte viability and integrity after low serum induction of differentiation. Therefore, the phenotypic rescue of dystrophy after deflazacort (DFZ) administration may result from a repression or limitation of JNK1 activity. To address this, we initially tested the DFZ response in nondystrophic myoblasts in which the only perturbation was elevated JNK1 activity. C2C12/MKK7 myoblasts were treated with a range of DFZ concentrations. Addition of DFZ to the medium (10 µg/mL) substantially increased the expression of differentiation-specific markers such as myosin heavy chain (MHC) compared with untreated C2C12/MKK7 myoblasts, and maintained differentiated myocyte viability and MHC expression 5 days after low serum induction (see Fig. 1 ). Nevertheless, DFZ treatment did not lead to a decrease in JNK1 phosphorylation.

View larger version (17K): [in this window] [in a new window]   Figure 1. Deflazacort (DFZ) can induce calcineurin activation in vitro. A) C2C12/MKK7 cultures have few myosin heavy chain (MHC) positive myoblast/myocytes after 72 h in serum-deprived medium (5%). Addition of the glucocorticoid prednisone to the medium increases this number to 12% (P<0.05). DFZ-treated cultures exhibit a further increase in differentiated myocyte viability as evidenced by the 20% MHC positive index. IGF-1, a known mobilizer of calcium, maintained differentiated myocyte viability to levels approximating that of prednisone-treated cells. Addition of both DFZ and the calcineurin inhibitor cyclosporin A (CsA) to the medium reduced differentiated myocyte viability to baseline levels, suggesting a direct link between the drug and calcinuerin activation. B) Calcineurin activity assay of C2C12/MKK7 cultures treated with DFZ alone or in combination with RU486 or CsA. Addition of DFZ resulted in a significant increase in free phosphate in the assay, a measure of calcineurin activity. Addition of DFZ and RU486 (10–8 M), a potent glucocorticoid receptor antagonist, produced a significant decrease in phosphate release, indicating a drop in calcinueirn activity. Similarly, DFZ administered with CsA (1.0 ng/mL) produced a decrease in calcineurin activity.

The ability of DFZ to limit the atrophic effect of JNK1 yet not perturb JNK1 activity appeared paradoxical. However, the corrective effects of DFZ could be explained by the premise that DFZ treatment may circumvent JNK1, activating the pathway/protein, which is inhibited by this kinase. A candidate is the calcineurin/NF-AT pathway. This pathway is impeded in dystrophic muscle by JNK1: JNK1 phosphorylates NF-ATc1, leading to nuclear export of this transcription factor. To examine the role of calcineurin in DFZ-induced rescue, C2C12/MKK7 cultures were exposed to DFZ and the calcineurin inhibitor cyclosporine (CsA). Treatment with DFZ and CsA, resulted in a dramatic decrease in differentiated myocyte viability and MHC expression, suggesting that inhibition of calcineurin was sufficient to block any rescue normally associated with DFZ (see Fig. 1 ). We then measured calcineurin activity directly in C2C12/MKK7 cultures. Besides DFZ, cultures were treated with DFZ plus RU486, an inhibitor of glucocorticoid receptor activity, and DFZ plus CsA. Addition of DFZ to the medium produced a significant up-regulation of calcineurin activity, with levels remaining constant over the time course measured. Cultures exposed to DFZ and CsA showed a decrease in calcineurin activity, as did a combination of DFZ plus RU486 (see Fig. 1 ). It is clear that DFZ treatment increases the enzymatic activity of calcineurin.

2. Deflazacort stimulates NF-ATc1 nuclear translocation and transcriptional activity in vitro
We examined the effect of DFZ on the probable calcineurin target NF-ATc1. In untreated C2C12/MKK7 cultures, NF-ATc1 accumulates in the cytoplasm. DFZ treatment of C2C12/MKK7 myocyte cultures revealed a change in NF-ATc1 localization, with equal distribution between cytosolic and nuclear compartments. DFZ treatment of C2C12/MKK7 myocytes resulted in an up-regulation of NF-ATc1-dependent activity as evidenced by the significant transcriptional activation of NF-AT-dependent promoter elements, including utrophin, IL-2, and IL-4. Together, these data indicate that DFZ counteracts the atrophic effects of JNK1 by activating the phosphatase calcineurin and restoring the nuclear localization and transcription activity of NF-ATc1.

3. Deflazacort treatment limits dystrophic myofiber degeneration in vivo by activation of the calcineurin pathway and up-regulation of utrophin expression
We examined the effects of DFZ treatment on dystrophic mouse skeletal muscle using the mdx strain and to see whether activation of the calcineurin/NF-AT pathway mediated the phenotypic rescue typically observed with glucocorticoid treatment. Three-wk-old mdx mice were subject to a variable treatment regime with vehicle (saline), DFZ, or DFZ and CsA injections. As expected, DFZ treatment alone largely inhibited the degenerative changes in the mdx skeletal muscle as evidenced by a 2-fold reduction in the number of centrally located myonuclei (hallmark of regenerating fibers), a reduction in the fiber size variability, and reduced evidence of degenerating fibers (Fig. 2 ). When mice were treated concurrently with CsA and DFZ, the phenotypic rescue of the myofibers was largely abolished, i.e., with more visible fiber damage, increased central nucleation, and increased fiber size variability vs. DFZ alone (see Fig. 2 ). These results reinforce the concept that DFZ acts directly on the dystrophic myofiber to limit disease progression.

View larger version (52K): [in this window] [in a new window]   Figure 2. Deflazacort-mediated activation of calcineurin attenuates the dystrophic phenotype in vivo. Histological representation using hematoxylin and eosin stain (H&E) of diaphragm muscle from A) saline-injected, B) DFZ-injected, and C) DFZ and cyclosporine (CsA) -treated mdx mice. D) Proportion of centrally nucleated fibers from diaphragm of mdx mice as described in panels A–C. The reduction in myofiber central nucleation demonstrates that damage is attenuated in DFZ-treated mice, which is reversed by simultaneous administration of CsA. (error=SEM, saline: n=5, DFZ: n=3, DFZ+CsA: n=3). E) Relative variability in the size of fiber cross-sectional area was compared from diaphragm of mdx mice as described in panels A–C. A greater variability in fiber size is representative of ongoing myofiber degeneration/regeneration (error bars=SEM, saline: n=3, DFZ: n=3, DFZ+CsA: n=3).

DFZ restoration of the calcineurin/NF-AT pathway suggested that an increase in NF-AT-mediated gene expression contributed to the ability of this drug to limit dystrophic muscle damage. Therefore, expression of the NF-AT-sensitive gene utrophin was monitored in dystrophic mdx mice subject to saline, DFZ, or DFZ and CsA treatment. DFZ treatment led to a significant increase in both utrophin gene expression and sarcolemmal localization of the utrophin protein itself. Moreover, the DFZ-dependent increase in utrophin expression was eliminated when dystrophic mice were subject to CsA injections. These data suggest that DFZ treatment limits dystrophic pathology by up-regulating utrophin expression via an increase in calcineurin activity, thereby maintaining the health of the existing myofiber population (Fig. 3 ).

View larger version (20K): [in this window] [in a new window]   Figure 3. Schematic representation of signaling perturbations in dystrophic muscle. In the absence of DFZ, elevated JNK activity in dystrophic muscle leads to NF-AT phosphorylation and removal from the nucleus (A). DFZ treatment leads to increased calcineurin activity, relocalization of NF-AT, and subsequent elevation in the expression of sarcolemmal utrophin (B).

CONCLUSIONS AND SIGNIFICANCE

Treatment of muscular dystrophy has focused on gene therapy approaches to redress the paucity of dystrophin protein and restore myofiber integrity. Nevertheless, elucidation of the mechanisms that underlie glucocorticoid relief of dystrophic progression has largely been ignored.

Our observations suggest that DFZ administration counteracts the negative signal influence of JNK-1 activity, although this effect does not involve manipulation of the JNK-1 kinase itself. Rather, DFZ treatment leads to the activation of calcineurin, which in turn provides the mechanism to dephosphorylate NF-ATc1, leading to its relocation and restoration of appropriate muscle gene expression (Fig. 3) . A common assumption in regard to glucocorticoid treatment of dystrophy is that glucocorticoids act to alleviate dystrophic pathology primarily through immunosuppression and reducing attendant inflammation. Although the data from this study do not confirm or deny this supposition, we did observe that a primary target of DFZ treatment appears to be skeletal muscle itself. Indeed, the increase seen in expression of the dystrophin homologue utrophin strongly argues in favor of a myofiber-sparing hypothesis. Up-regulation of utrophin expression has been demonstrated to provide a functional correction for dystrophin deficiency and the additional absence of utrophin has been shown to accelerate dystrophy pathogenesis. Therefore, myofiber protection is a key outcome for DFZ efficacy in dystrophic muscle.

Despite the obvious benefits to accrue from DFZ up-regulation of utrophin expression, the restorative effect of this drug may depend on other NF-AT-sensitive targets. For example, we have observed that the NF-AT-responsive IL-4 promoter is activated during DFZ treatment of muscle cell cultures and is as robust as that observed for utrophin. IL-4 itself has been shown to promote the fusion of myoblasts to preexisting myotubes. Although we not directly test the requirement of IL-4, it is reasonable to suggest that the full range of DFZ effects may include an IL-4-mediated increase in myoblast fusion during ongoing degeneration and regeneration of the dystrophic muscle life cycle.

Observations from the current study indicate that an effective treatment for dystrophy patients may finally be realized with the use of combinatorial pharmacologic strategies and rationale drug design, selectively targeting an increase in calcineurin activity, and/or inhibiting JNK1 activity.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1859fje;

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