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The effect of glucocorticoids on the accumulation of utrophin by cultured normal and dystrophic human skeletal muscle satellite cells
Neuromusc.Disord.,Vol.5. No. 2, pp. 105-114, 1995 ElsevierScienceLtd Printedin Great Britain 0960-8966/95 $9.50 + 0.00
Pergamon
0960--8966(94)00042--5
THE EFFECT OF GLUCOCORTICOIDS ON THE ACCUMULATION OF UTROPHIN BY CULTURED NORMAL AND DYSTROPHIC HUMAN SKELETAL MUSCLE SATELLITE CELLS F. PASQUINI,* C. GUERIN,* DL B L A K E , t K. DAVIES,t G. KARPATI* and P. HOLLAND*:~ *Neuromuscular Research Group, Montreal Neurological Institute, 3801 University Street, Montreal, QC, Canada H3A 2B4; tMolecular Genetics Group, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK
(Received 14 January 1994; revised 11 May 1994," accepted 2 June 1994)
Abstract--Human muscle cultures undergo a long-term loss of myotubes and a decline in dystrophin content, which can be prevented by glucocorticoid treatment of the cultures. We confirmed these findings and extended them to show that the utrophin content of control and dexamethasone-treated normal myotube cultures is not significantly different. In contrast to normal cultures, the utrophin content of long-term dexamethasone-treated DMD myotube cultures was significantly greater than that of the corresponding untreated cultures. Utrophin mRNA transcript levels normalized to total poly (A) were unaffected by dexamethasone treatment of either normal or DMD myotube cultures, suggesting the effect of dexamethasone on utrophin accumulation by DMD cultures is mediated post-transcriptionally. A combination of an increase in myotube numbers and lack of competition with dystrophin for membranebinding sites in DMD myotubes may explain the distinct effects of dexamethasone on utrophin levels in normal and DMD cultures. Key words: Utrophin, myogenesis, prednisone
dystrophin, glucocorticoids,
INTRODUCTION
Clinical trials have shown that the glucocorticoid prednisone has a long-lasting beneficial effect on muscle strength and function in Duchenne muscular dystrophy (DMD) patients [1, 2]. The molecular basis of this beneficial effect is not understood. Glucocorticoids exert a variety of effects on muscle metabolism, growth and development [3, 4]. Although certain of these effects are catabolic in nature (e.g., elevation of protease activity) positive stimulatory effects of glucocorticoids on myoblast proliferation and differentiation have been reported [5, 6]. Recent studies have examined the effect of glucocorticoids on the myogenesis of normal
~Author to whom correspondence should be addressed at: Muscle Biochemistry Laboratory, Room 665, Montreal Neurological Institute, 3801 University Street, Montreal, QC, H3A 2B4, Canada.
Duchenne muscular dystrophy,
and dystrophic muscle in vitro. The addition of ot-methylprednisolone to differentiating normal human muscle satellite cell cultures increased their content of dystrophin and myosin heavy chain [7]. This effect was attributed to inhibition of myotube death, rather than to a specific stimulation of dystrophin expression per myotube nucleus. A later study did, however, obtain some evidence indicating a specific effect of amethylprednisolone on dystrophin accumulation. Some clones established from normal muscle and most clones established from biopsies of D M D or Becker muscular dystrophy (BMD) patients, showed impaired myogenesis on exposure to o~-methylprednisolone [8]. In such clones, dystrophin accumulation was greater than in parallel, untreated, cultures [9]. Stimulation of dystrophin expression may therefore be relevant to glucocorticoid action in BMB muscle, or in those cases of D M D where some functional dystrophin is expressed. 105
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Obviously such an effect cannot explain how glucocorticoids are beneficial in cases of D M D in which no functional dystrophin is expressed. The reported effect of o~-methylprednisolone on dystrophin expression raises the possibility that glucocorticoids may also affect the expression of the dystrophin homologue, utrophin. Utrophin and dystrophin are of similar size and are homologous along their entire length, suggesting they derive from a common ancestral gene [10]. Unlike dystrophin, which is restricted in its expression to differentiated skeletal muscle, heart and brain, utrophin is expressed in a variety of fetal and adult tissues. Utrophin is, however, present at a low concentration in normal adult skeletal muscle [11], where it is restricted to the neuromuscular junction [12] and vascular components of smooth muscle [14]. In adult dystrophin-deficient muscle, utrophin is more abundant than normal and is present throughout the sarcolemma [13-15]. Assuming utrophin has a similar function to dystrophin, upregulation of utrophin expression might partially compensate for dystrophin deficiency [13, 14]. The possibility that utrophin might functionally compensate for dystrophin is supported by the recent observation that both of these proteins specifically associate with the same sarcolemmal glycoprotein complex [15]. To date the effect of glucocorticoid treatment on utrophin expression in human muscle cells has not been examined, a-Methylprednisolone has, however, been reported to enhance the accumulation of utrophin in primary cultures of mdx mouse muscle [16]. In the present study we show that dexamethasone-treated D M D muscle cultures have a higher utrophin level than control untreated D M D cultures. Interestingly, the accumulation of utrophin by normal muscle cultures was not significantly increased by dexamethasone treatment. We further show that the effect on D M D muscle cultures must be mediated post-transcriptionally, since dexamethasone has no effect on utrophin mRNA transcript levels. MATERIALS AND METHODS
Cell culture Satellite cells were obtained from biopsies of human biceps muscle taken from typical D M D
patients or, as controls, from patients with suspected neuromuscular disease, who on subsequent histological analysis had no detectable muscle pathology. The biopsies were treated with trypsin and collagenase to liberate satellite cells as previously described [17]. Satellite cells were purified by fluorescence-activated cell sorting (FACS) [18] and were grown in Ham's F12 medium supplemented with 15% fetal calf serum and Ham's serum-free supplement without dexamethasone [19]. Fresh growth medium was added every three days. At confluence the cells were switched to a medium low in serum (Dulbecco's modified Eagle's medium containing 2% horse serum) to increase differentiation. When present, dexamethasone was added at the time of the switch to DMEM, 2% horse serum. This medium was also renewed every three days until the cultures were harvested. Electrophoresis and western blotting Cultures were harvested by scraping into modified Laemmli sample buffer [20] containing 15% (w/v), SDS, preheated to 100°C and were solubilized by immediate boiling for 5 rain. Gel electrophoresis and western blotting were essentially as previously described [20, 21]. Samples corresponding to 12 Ixg of total cellular protein were analysed by SDS/PAGE on a 5.5% acrylamide, 0.75 cm thick separating gel. The gel was equilibrilated in transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3, 20% (v/v) methanol) for 15 min and electroblotted to nitrocellulose for 2 h at 100 V with cooling. Blots were rinsed with water, air dried and blocked by incubation for 40 rain in 10% skim milk in TBST (0.9% NaC1, 0.1% Tween-20, 50 mM Tris-HC1, pH 7.5). The blots were incubated for 3 h at room temperature with antibodies to dystrophin [22] or utrophin [13] at a 1 : 1000 dilution and for 1 h with second antibodies, using the ECL system (Amersham). Immunoreactive bands corresponding to dystrophin or utrophin were quantitated by video densitometry using the JAVA software program of Jandel Scientific and the values were normalized to total protein content of the cultures, determined by a modified Lowry procedure [23]. For normal cultures, utrophin and dystrophin were probed on samples from the same culture dishes. This provided an internal control for cases where dexamethasone had an effect on dystrophin accumulation, but no
Glucocorticoids and Utrophin
effect on utrophin accumulation. For analysis of the effect of different doses of dexamethasone, if the number of samples exceeded the capacity of a single blot, a set of common samples was included as an internal reference standard on each blot.
Statistical analysis of data The effect of various doses of dexamethasone on utrophin and dystrophin levels was first analysed by one way analysis of variance (ANOVA). This revealed a statistically significant effect (P<0.05) of dexamethasone on dystrophin levels in normal cells (P -- 0.003) and on utrophin levels in DMD cells (P = 0.019) but not on utrophin levels in normal cells (P = 0.8). Further analysis by ANOVA of individual group means using the Bonferroni method revealed no statistically significant differences between the effect of various doses of dexamethasone (0.1-50 ixM) on utrophin or dystrophin accumulation. Utrophin and dystrophin levels measured at all dexamethsone doses were therefore combined and compared with control values by the unpaired t-test (two tailed). This is shown in Fig. 5. The P values obtained were P<0.0001 for dystrophin in normal cells (sample number of nine for controls and 59 for dexamethasone-treated); P<0.0001 for utrophin in DMD cells (sample number of six for controls and 39 for dexamethasone-treated); P = 0.28 for utrophin in normal cells (sample number of six for controls and 38 for dexamethasone-treated).
Antibodies The antibody used to detect dystrophin was a rabbit polyclonal antibody raised against a synthetic peptide corresponding to the C-terminal 17 amino acid residues of the published human dystrophin sequence [24]. Antibodies were affinity purified before use using Affi-Gel (BioRad) conjugated to the synthetic peptide. We have previously shown this antibody does not recognize utrophin in immunoblots or in immunocytochemistry [14]. The antibody used to detect utrophin was the generous gift of Dr G. E. Morris. It is a mouse monoclonal antibody raised against a [3-galactosidase fusion protein containing 1.05 kb of sequence encoding the C-terminal domain of utrophin. The antibody has been termed MANCHO7 and has been previously shown
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not to cross-react with dystrophin in immunoblots or in immunocytochemistry [13].
Quantitation of myoblast fusion and myotube number Sister culture dishes to those used for dystrophin and utrophin analysis were rinsed with PBS, fixed with 2.5% glutaraldehyde and stained with haematoxylin and eosin. For each plate, total nuclei, nuclei in myotubes, and number of myotubes per field were counted in nine random fields at 400 × magnification. Only cells containing more than two nuclei were counted as myotubes.
Isolation of mRNA and northern blotting Polyadenylated mRNA was purified from cultured cells using the Pharmacia Quick Prep Micro mRNA purification kit. The RNA species were resolved in 1% agarose gels containing 3.7% formaldehyde. The fractionated RNA species were then blotted onto nylon membranes (Hybond-N, Amersham) by overnight capillary transfer. Blots were incubated overnight at 42°C in prehybridization buffer composed of 45% deionized formamide, 5 × SSC (20 x SSC consists of 3 M NaC1, 0.3 M sodium citrate pH 7), 500 mM sodium phosphate buffer pH 6.8, 10 x Denhardt's solution (100 × Denhardt's solution consists of 2% each of Ficoll 400, polyvinylpyrolidone, and BSA), 0.5 mg ml heat denatured salmon sperm DNA and 0.5% SDS. Blots were then incubated in hybridization buffer, containing radiolabelled cDNA probe. The hybridization buffer was composed of 50% deionized formamide, 5 × SET (20 × set consists of 3M NaCI, 0.6 M Tris-HCL pH 8, 40 mM EDTA), 1 x Denhardt's solution (as above), 0.1 mg ml1 heat denatured salmon sperm DNA and 0.5% SDS. The cDNA probes used were an 873 bp fragment (position 7002 7875) of dystrophin cDNA and a 992 bp fragment (position 6771-7703 of the EMBL sequence) of utrophin cDNA. These probes were heat denatured before addition to the hybridization buffer. Blots were incubated with the radiolabelled cDNA probe overnight at 42°C and were then washed extensively in 0.1 x SSC, 0.1% SDS buffer for 2.5 h at 65°C and subjected to autoradiography and phosphorimaging (PhosphorImager, Molecular Dynamics). Bands were quantitated using the
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ImageQuant software program (Molecular Dynamics). All mRNA levels were normalized relative to the total hybridization signal per lane obtained on probing the blot with synthetic poly U, end-labeled with [~.32p] ATP using T4 polynucleotide kinase. Hybridization conditions for poly U were as described above but at 25°C and washing was with 2 × SSC, 0.1% SDS for 1 h at 65°C. RESULTS
Effect of dexamethasone on myogenesis Both normal and D M D cultures fused extensively within 3-5 days following a switch from growth medium to serum-depleted fusion medium (Fig. 1). The initial fusion capacity of the satellite cell cultures was not affected by dexamethasone (Fig. 1, panels B and D) and was similar for normal (Fig. 1, panels A and B) and D M D (Fig. 1, panels C and D) cultures. The average % nuclei in myotubes after three days in fusion medium was 52 + 7% for normal
untreated cultures; 50 + 7% for normal dexamethasone-treated cultures; 66 + 12% for untreated D M D cultures and 61 _+ 3% for dexamethasone-treated D M D cultures. These values are close to the average final proportion of total nuclei found in myotubes of 61 + 5% reported by Blau and Webster [25] for clonal human muscle cell cultures. As originally reported by Sklar and Brown for normal cultures [7], if the cultures were subsequently maintained for a prolonged period in the absence of dexamethasone there was a gradual decline in the number of myotubes per area, starting 4-5 days after the switch to fusion medium. Table 1 shows that 15 days following the switch of D M D cells to fusion medium few myotubes remained in untreated cultures. In contrast if the cultures were maintained in fusion medium supplemented with dexamethasone, myotube survival was dramatically increased, with the optimal dose being in the range of 0.2-1.0 IxM dexamethasone. In this dose range the % nuclei in myotubes (-50%) remained close to that observed following the
Fig. 1. The effect of dexamethasone on the fusion of normal and DMD satellite cells. Satellite cells from normal human muscle (panels A and B) and from muscle of DMD patients (panels C and D) were grown to confluence and switched to fusion medium with (panels B and D) or without (panels A and C) 1 p.M dexamethasone. Cells were fixed and stained three days later. Representative fields photographed at 400 × magnification are shown. Note the presence of large multinueleate myotubes in all four panels. Quantitation of the percentage of total nuclei in myotubes in nine such randomly selected fields for each set of cells revealed no apparent effect of dexamethasone on the initial fusion of normal or DMD cultures (see text).
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Glucocorticoids and Utrophin Table 1. The effect of dexamethasone on DMD satellite cell fusion and density of myotubes Dexamethasone (~M) Experiment i 0.0 0.1 0.2 0.5 1.0 2.0 5.0 10.0 50.0 Experiment 2 0.0 0.1 0.2 0.5 1.0 5.0 10.0 50.0
Total nuclei
Nuclei in myotubes
Number of myotubes
% Nuclei in myotubes
263 294 342 228 333 268 230 233 223
+24 "+34 _+36 +_24 + 26 -+38 + 30 _+ I1 -+33
12 _+ 11 38 _+ 18 93 -+ 32 117 _+ 18 107 ± 12 67 "+20 51 +,%12 45 -+9 29 _+7
0.4 3.0 5.6 9.2 8.6 5.4 5.9 5.1 3.6
"+0.4 _+ 1.4 _+ 1.7 _+ 1.5 -+ t.0 _+ 1.3 -+ 1.2 + 1.0 +0.8
4.4 12.8 27.2 51.3 32.3 25.1 20.3 19.2 13.0
223 237 141 203 225 202 215 171
_+ 16 _+ 15 _+ 13 + 33 _+ 16 -+ 20 _+ 15 + 17
2 _+ 1 71 + 15 75 + 12 100 +_25 75 -+ 10 67 _+ 11 49 _+5 49 -+ 10
0.3 7.4 8.3 8.8 7.6 6.7 4.9 5.1
_+0.2 + 1.1 + 1.2 _+ 15. -+ 1.0 -+ 1.0 +_0.4 "+0.7
1.1 30.0 53.3 49.2 33.4 33.3 22.8 28.6
Confluent cultures were maintained for 15 days in fusion medium supplemented with the indicated concentrations of dexamethasone. The cells were fixed with glutaraldehyde and stained with haematoxylin and eosin. Nuclei and myotubes were counted in nine randomly selected fields at 400 × magnification. Values are mean _+ S.E.M. per field.
initial period of myotube formation. Similar results were obtained for normal cells (not shown).
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Effect of dexamethasone on dystrophin accumulation When normal cultures were grown and maintained in medium with or without 1 p~M dexamethasone, dystrophin levels were maintained in supplemented cultures, but declined in cultures which were continuously maintained in unsupplemented medium (Fig. 2, panel A). This confirms an earlier report of a positive effect of ot-methylprednisolone at this concentration on dystrophin accumulation by cultured human muscle cells [7]. As expected, no immunoreactive dystrophin band was detectable in D M D cultures (Fig. 2, panel B).
Utrophin accumulation by normal and DMD cultures Utrophin levels in normal cells appeared essentially unaffected by long-term dexamethasone treatment (Fig. 3, panel A). In contrast, in D M D cultures, utrophin was maintained at a higher level in dexamethasone-treated cells (Fig. 3, panel B).
Dose response We next tested the effect of various doses of dexamethasone on dystrophin and utrophin accumulation by normal and D M D cells. Such
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Fig. 2. The effect o f d e x a m e t h a s o n e o n the m a i n t e n a n c e o f d y s t r o p h i n levels in n o r m a l satellite cell cultures. The figure shows a w e s t e r n blot o f 12 i~g samples of t o t a l p r o t e i n f r o m n o r m a l m u s c l e cultures (panel A) a n d D M D m u s c l e cultures (panel B) m a i n t a i n e d for seven d a y s (lanes 1 & 2) a n d 15 d a y s (lanes 3 a n d 4) in fusion m e d i u m w i t h (+) or w i t h o u t (-) 1 p,M d e x a m e t h a s o n e , Blots were incub a t e d w i t h a n t i b o d i e s specific for the C - t e r m i n a l 17 a m i n o acids o f d y s t r o p h i n . T h e m a j o r i m m u n o r e a c t i v e b a n d h a d a n a p p a r e n t m o l e c u l a r w e i g h t o f - 4 0 0 k D a (arrow, p a n e l A) a n d was n o t a b l y greater in a m o u n t in d e x a m e t h a s o n e t r e a t e d cultures b y 15 d a y s after the switch to fusion m e d i u m . N o i m m u n o r e a c t i v e d y s t r o p h i n b a n d was d e t e c t a b l e in D M D cultures (panel B),
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different doses of dexamethasone used, all data obtained at the various doses were combined for further statistical analysis. Figure 5 shows that 0.2-50 p~M dexamethasone had a significant effect on utrophin accumulation by D M D cells and on dystrophin accumulation by normal cells, whereas utrophin accumulation by normal cells was not significantly affected.
Effect o f dexamethasone on m R N A transcript levels
B
To determine whether the effects of dexamethasone on utrophin accumulation may be mediated transcriptionally, m R N A was prepared from normal and D M D cultures which had been treated for either 24 h or for 5 days with dexamethasone. We deliberately used + + shorter times of treatment with dexamethasone day 7 day 15 in these experiments than were used previously Fig. 3. The effect of dexamethasone on utrophin accumula- for studies at the protein level. Our rationale tion by normal and DMD cultures. Western blot of 12 p~g was that a significant direct effect of dexasamples of total protein from normal (panel A) and DMD methasone on transcription would be detect(panel B) muscle cultures maintained for seven days (lanes 1 and 2) and 15 days (lanes 3 and 4) in fusion medium with able over this shorter period. We also wished to (+) or without (-) 1 ~M dexamethasone after reaching discriminate such an effect from indirect elevaconfluence. Blots were incubated with antibodies specific for utrophin. The major immunoreactive band had an tion of m R N A levels due to myotube rescue. apparent molecular weight of ~400 kDa and was notably Table 2 shows that neither treatment increased greater in amount in dexamethasone-treated DMD cultures the accumulation of utrophin m R N A after than in the corresponding control cultures by 15 days after the switch to fusion medium (panel B, lanes 3 and 4) but normalization for total polyadenylated m R N A present in the blot. An apparent stimulatory not in normal cultures (panel A, lanes 3 and 4). effect on dystrophin m R N A accumulation was detected. an experiment is illustrated in Fig. 4. In this experiment all cultures were harvested 15 days DISCUSSION after the switch to fusion medium, with or without added dexamethasone. Each lane represents a sample from a different culture dish. It can be seen that dystrophin levels in normal cells (panel A) and utrophin levels in D M D cells (panel C) were reproducibly higher than in controls at all doses of dexamethasone tested. In contrast utrophin levels in normal cultures were not reproducibly affected by dexamethasone treatment (panel B). The immunoreactive bands obtained at doses ranging from 0.1 to 50 ~ M dexamethasone were quantitated by video densitometry. Statistical analysis (see Materials and Methods) of the data obtained revealed a significant effect of dexamethasone on dystrophin levels in normal cultures and on utrophin levels in D M D cultures but no significant effect on utrophin levels in normal cultures. Since no statistically significant differences in utrophin or dystrophin levels were detected between the
In this study we have examined the effect of treatment o f confluent normal and D M D satellite cell cultures with dexamethasone following the switch of confluent cultures to a low-serum, fusion-promoting, medium. N o r m a l and D M D cultures responded differently. In D M D cultures a marked effect on utrophin level was observed. In contrast, in normal cultures no significant effect of dexamethasone on utrophin was detectable, although the same treatment markedly increased their dystrophin content. The effects of dexamethasone on the dystrophin content of normal cultures and the utrophin content of D M D cultures were not due to an effect on the initial stages of terminal differentiation of the cultures as they were only evident after several days of dexamethasone treatment (Figs. 2 and 3). Sklar and Brown [7] were the first to show
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Glucocorticoids and Utrophin
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7 8
9 10 11 12
A -dys
B - -utr i i~iii i~ ~ i
~ ~
~
!il
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-utr
Fig. 4. The effect of various doses of dexamethasone on utrophin and dystrophin levels in normal and DMD muscle cultures. Normal (panels A and B) and DMD (panel C) muscle cultures were harvested after 15 days in fusion medium supplemented with the indicated concentrations of dexamethasone. The figure shown represents western blots of samples (12 Itg) of total protein incubated with antibodies specific for dystrophin (panel A) and utrophin (panels B and C). Arrows at the right of each panel represent the position of the major ~400 kDa immunoreactive band (dys = dystrophin; utr = utrophin). Panels A and B~lanes 1 and 2, 0 I~M dexamethasone; lanes 3 and 4, 0.2 ~,M dexamethasone; lanes 5-7, 0.5 ~M dexamethasone; lanes 8 and 9, 1.0 p,M dexamethasone; lanes 10-12, 2.0 p,M dexamethasone. Panel C--lanes 1-4, 0 p.M dexamethasone; lanes 5-8, 0.1 IxM dexamethasone; lanes 9-11, 0.2 txM dexamethasone; lane 12, 0.5 ~M dexamethasone.
that glucocorticoids can increase the dystrophin content of human satellite cell cultures by inhibiting the naturally occurring death of mature myotubes. In the present study we show a similar long-term effect of dexamethasone on utrophin accumulation by DMD muscle cultures. This effect was associated with a greater number of myotubes present in DMD cultures maintained long-term in dexamethasone-supplemented fusion medium. Taken together these findings suggest that rescue of myotubes by glucocorticoid treatment may also be a significant factor in the observed stimulatory effect of dexamethasone on utrophin accumulation by DMD satellite cell cultures. It should be noted, however, that the correlation with myotube rescue was not perfect. Dexamethasone had a marked biphasic dose-depen-
dent effect on myotube survival (Table 1) over the range 0.1-50 IxM, whereas no significant differences were noted between different doses over this range for the dexamethasone effect on utrophin or dystrophin accumulation. Dystrophin is a muscle-specific protein which is known to increase during myoblast differentiation [26], it can therefore readily be seen how rescue of mature myotubes from cell death could cause a relative increase in dystrophin accumulation. The mechanism by which myotube rescue could increase the relative content of utrophin is less obvious. Utrophin is not muscle specific and does not increase in muscle cultures as a consequence of differentiation [14]. Passaquin et al. [16] have recently reported that the glucocorticoid ot-methylprednisolone
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Utrophin (Duchenne)
Fig. 5. Quantitative analysis of the effect of dexamethasone on utrophin and dystrophin levels in normal and D M D satellite cell cultures. Normal and D M D satellite cells were maintained for 15 days in fusion medium with (hatched bars) or without (open bars) 0.1-50 ~M dexamethasone. Cells were harvested and 12 ~g samples were immunoblotted as described for Fig. 4. The ~400 kDa immunoreactive bands were quantitated by video densitometry. The values shown represent mean + S.E,M. Further details of the statistical analysis are given in the Materials and Methods section. The asterisks above the hatched bars indicate a statistically significant increase in dystrophin or utrophin levels, compared to the untreated controls.
increases the utrophin content of m d x mouse muscle cultures. A similar protocol was used to that in the present study, in that the glucocorticold was added at the onset of myoblast fusion. In the m d x study an effect on utrophin accumulation was evident after seven days of a-methylprednisolone treatment and this correlated with a stimulatory effect on myotube numbers. The antibody used to detect utrophin in the m d x studies cross-reacted with dystrophin, making it impossible to tell whether c~methylprednisolone also stimulated utrophin accumulation in normal mouse muscle.
Collectively, the results of this study and the study of Passaquin et al. [16] clearly show that glucocorticoid treatment of dystrophindeficient human and mouse muscle cultures, respectively, can enhance their content of utrophin relative to other cellular proteins. The question remains whether this is due to stimulation of transcription, or increased stability of mRNA or protein. Two other findings in the present study provide further insight into the mechanism underlying this effect. First is the observation that there was no detectable effect of dexamethasone on utrophin mRNA transcript levels. This suggests that the effects of dexamethasone on utrophin accumulation are mediated post-transcriptionally. Interestingly, we did obtain some indication of a stimulatory effect of dexamethasone on dystrophin mRNA accumulation. Further studies are required to demonstrate the reproducibility of this effect. A positive effect of dexamethasone on dystrophin mRNA levels would support recent studies at the protein level [9,16] which suggest that glucocorticoids may selectively effect dystrophin expression in cultured muscle cells. As discussed by Hardiman et al. [9] the muscle promoter for dystrophin does not contain a glucocorticoid response element [27] indicating that a transcriptional effect of dexamethasone on dystrophin expression would have to be indirect or be mediated via an alternate promoter, such as the brain promoter. It has not yet been established whether any of the dystrophin promoters have a glucocorticoid response element. Secondly, we observed that the effect of dexamethasone on utrophin accumulation was
Table 2. The effect of dexamethasone on utrophin and dystrophin mRNA levels in normal and DMD satellite cell cultures Dexamethasone treatment Normal cells DMD cells
None 24 hr 5 days None 24 hr 5 days
Dystrophin/poly A
Utrophin/poly A
1.0 1.4 1.7 n.d. n.d. n.d.
1.0 0.8 1.1 1.0 0.9 1.1
Confluent cultures were grown under three different conditions: (i) for five days in fusion medium without added dexamethasone; (ii) for four days in fusion medium without dexamethasone followed by 24 h in medium supplemented with 1 IxM dexamethasone; (iii) for five days in fusion medium supplemented with l IxM dexamethason. Cultures were harvested and mRNA was isolated and analysed by northern blotting. Values shown are the mean of two separate experiments and are normalized to poly (A) mRNA, with the control values arbitrarily set at 1.0. Note that normalization to measured poly (A) levels indicates dexamethasone did not selectively alter utrophin mRNA levels.
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much more dramatic in DMD cultures than it was in normal cultures. Any mechanism proposed to explain the effect of dexamethasone on utrophin accumulation by D M D cells must take into account this relative lack of effect of dexamethasone on utrophin accumulation by normal cells. Furthermore, regardless of the explanation of this observation, it clearly indicates that the effect of dexamethasone on utrophin accumulation in D M D cells is largely a secondary consequence of dystrophin deficiency. One possible mechanism is suggested by the recent observation that utrophin and dystrophin bind to the same sarcolemmal glycoprotein complex [15]. Given this fact, it is tempting to speculate that utrophin may be more stable in D M D myotubes than in normal dystrophin-containing myotubes, because in D M D cells utrophin does not have to compete with dystrophin for binding to the dystroglycan complex. According to this model, utrophin bound to the dystroglycan complex would be less susceptible than unbound utrophin to protein turnover and would therefore accumulate to a greater extent within the myotube. Dexamethasone, by rescuing mature myotubes from cell death, would therefore increase the relative utrophin content of DMD cultures over that of untreated cultures. This model of increased accumulation of utrophin in D M D myotubes compared to normal myotubes as a secondary consequence of decreased competition with dystrophin is also consistent with the observation of increased accumulation of utrophin in D M D and BMD muscle [14, 28]. Marked up-regulation of utrophin in extraocular muscle and cardiac muscle of the mdx mouse has been shown to be associated with a corresponding increase in the 50 and 156 kDa components of the dystrophin/glycoprotein complex [15] providing further evidence of an interaction between utrophin and the dystroglycan complex in vivo. A further factor which could contribute to the dexamethasone effect on utrophin, is that a large protein like utrophin may be more stable in a mature non-dividing cell like a myotube than it is in a dividing satellite cell, in which the membrane cytoskeleton is constantly being disassembled and reassembled during cell division. While such an effect would clearly favor utrophin accumulation in both normal and D M D cells, competition with dystrophin for
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membrane binding sites may counteract this benefit and lower the stability of utrophin in normal myotubes. In conclusion, although glucocorticoids may be beneficial in D M D by aiding the regeneration process (e.g., by increasing the survival of newly regenerant myotubes prior to innervation) and consequently stabilizing utrophin levels in mature myotubes, the present study indicates that they do not act directly to increase utrophin expression. are grateful to the Muscular Dystrophy Association of Canada, the Muscular Dystrophy Group of Great Britain, the Muscular Dystrophy Association (U.S.A.) and the Medical Research Council for financial support. F. P. was the recipient of a University of Toronto Medical Alumni Fellowship. We also thank Dr G. Morris for the MANCHO7 antibody and Dr J. Tinsley for assistance in isolation of the original cDNA clones used in this study. Acknowledgements--We
REFERENCES 1. Fenichel G M, Florence J M, Pestronk A, et al. Longterm benefit from prednisone in Duchenne muscular dystrophy. Neurology 1991; 41: 1874-1877. 2. Mendell J R, Moxley R T, Griggs R C, et al. Randomized, double-blind six-month trial of prednisone in Duchenne's muscular dystrophy. N Engl J M e d 1989; 320: 1592-1597. 3. Florini J R. Hormonal control of muscle growth. Muscle Nerve 1987; 10: 577-598. 4. Florini J R, Ewton D Z, Magri K A. Hormones, growth factors, and myogenic differentiation. Annu Rev Physiol 1991; 53: 201-216. 5. Florini J, Roberts S. A serum-free medium for the growth of muscle cells in culture. In vitro 1979: 15: 983-992. 6. Ball E, Sanwal B. A synergistic effect of glucocorticoids and insulin on the differentiation of myoblasts. J Cell Physiol 1980; 102: 27-36. 7. Sklar R M, Brown Jr R H. Methylprednisolone increases dystrophin levels by inhibiting myotube death during myogenesis of normal human muscle in vitro. J Neurol Sci 1991: 101: 73-81. 8. Hardiman O, Brown Jr R H, Beggs A H, Specht L, Sklar R M. Differential glucocorticoid effects on the fusion of Duchenne/Becker and control muscle cultures: pharmacologic detection of accelerated aging in dystrophic muscle. Neurology 1991: 42. 9. Hardiman O, Sklar R M, Brown Jr R H. Methylprednisolone selectively affects dystrophin expression in human muscle cultures. Neurology 1993: 43: 342-345. 10. Tinsley J M, Blake D J, Roche A, et al. Primary structure of dystrophin-related protein. Nature 1992: 360:591 593. 11. Love D R, Morris G E, Ellis J M, et al. Tissue distribution of the dystrophin-related gene product and expression in the m d x and dy mouse. Proc Natl Acad Sci USA 1991, 8g: 3243-3247. 12. Ohlendieck K, Ervasti J M, Matsumura K, Kahl S D, Leveille C J. Campbell K P. Dystrophin-related protein is localized to neuromuscular junctions of adult skeletal muscle. Neuron 1991: 7:499 508.
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13. Thi Man N, Ellis J M, Love D R, Davies K E, Gatter K C, Dickson G, Morris G E. Localization of the DMDL gene-encoded dystrophin-related protein using a panel of nineteen monoclonal antibodies: presence at neuromuscular junctions, in the sarcolemma of dystrophic skeletal muscle, in vascular and other smooth muscles, and in proliferating brain cell lines. J Cell Biol 1991; 115: 1695-1700. 14. Karpati G, Carpenter S, Morris G E, Davies K E, Guerin C, Holland P. Localization and quantitation of the chromosome 6-encoded dystrophin-related protein in normal and pathological human muscle. J Neuropathol Exp Neurol 1993; 52: 119-128. 15. Matsumura K, Ervasti J M, Ohlendieck K, Kahl S D, Campbell K P. Association of dystrophin-related protein with dystrophin-associated proteins in mdx mouse muscle. Nature 1992; 360: 588-591. 16. Passaquin A C, Metzinger L, L6ger J J, Warter J-M, Poindron P. Prednisolone enhances myogenesis and dystrophin-related protein in skeletal muscle cell cultures from mdx mouse. J Neurosei Res 1993; 35: 363-372. 17. Champaneria S, Holland P, Karpati G, Gu6rin C. Developmental regulation of cell-surface glycoproteins in clonal cultures of human skeletal muscle satellite cells. Bioehem Cell Biol 1989; 67: 128-136. 18. Webster C, Pavlath G, Parks D, Walsh F, Blau H M. Isolation of human myoblasts with the fluorescenceactivated cell sorter. Exp Cell Res 1988; 174: 252-265. 19. St Clair J A, Meyer-Demarest S D, Ham R G. Improved medium with EGF and BSA for differentiated human skeletal muscle cells. Muscle Nerve 1992; 15: 774-779. 20. Nicolson L. Dystrophin in skeletal muscle II. Immunoreactivity in patients with Xp21 muscular
dystrophy. J Neurol Sci 1989; 94: 137-146. 21. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350-4354. 22. Weller B, Karpati G, Lehnert S, Carpenter S, Ajdukovic B, Holland P. Inhibition of myosatellite cell proliferation by gamma irradiation does not prevent the age-related increase of the number of dystrophinpositive fibres in soleus muscles of mdx female heterozygote mice. Am J Pathol 1991; 138: 1497-1502. 23. Lowry O, Rosebrough N, Farr A, Randall R. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265-275. 24. Zubrzycka-Gaarn E, Bulman D, Karpati G, et al. The Duchenne muscular dystrophy gene product is localized in sarcolemma of human skeletal muscle. Nature 1988; 333: 466-469. 25. Blau H, Webster C. Isolation and characterization of human muscle cells. Proc Natl Acad Sci USA 1981; 78: 5623-5627. 26. Nudel U, Robzyk K, Yaffe D. Expression of the putative Duchenne muscular dystrophy gene in differentiated myogenic cell cultures and the brain. Nature 1988; 331: 635-638. 27. Klamut H J, Gangopadhyay S B, Worton R G, Ray P N. Molecular and functional analysis of the musclespecific promoter region of the duchenne muscular dystrophy gene. Mol Cell Biol 1990; 10: 193-205. 28. Helliwell T R, Nguyen thi Man, Morris G E, Davies K E. The dystrophin-related protein, utrophin, is expressed on the sarcolemma of regenerating human skeletal muscle fibres in dystrophies and inflammatory myopathies, Neuromuse Disord 1992; 2: 177-184.
Pergamon
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THE EFFECT OF GLUCOCORTICOIDS ON THE ACCUMULATION OF UTROPHIN BY CULTURED NORMAL AND DYSTROPHIC HUMAN SKELETAL MUSCLE SATELLITE CELLS F. PASQUINI,* C. GUERIN,* DL B L A K E , t K. DAVIES,t G. KARPATI* and P. HOLLAND*:~ *Neuromuscular Research Group, Montreal Neurological Institute, 3801 University Street, Montreal, QC, Canada H3A 2B4; tMolecular Genetics Group, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK
(Received 14 January 1994; revised 11 May 1994," accepted 2 June 1994)
Abstract--Human muscle cultures undergo a long-term loss of myotubes and a decline in dystrophin content, which can be prevented by glucocorticoid treatment of the cultures. We confirmed these findings and extended them to show that the utrophin content of control and dexamethasone-treated normal myotube cultures is not significantly different. In contrast to normal cultures, the utrophin content of long-term dexamethasone-treated DMD myotube cultures was significantly greater than that of the corresponding untreated cultures. Utrophin mRNA transcript levels normalized to total poly (A) were unaffected by dexamethasone treatment of either normal or DMD myotube cultures, suggesting the effect of dexamethasone on utrophin accumulation by DMD cultures is mediated post-transcriptionally. A combination of an increase in myotube numbers and lack of competition with dystrophin for membranebinding sites in DMD myotubes may explain the distinct effects of dexamethasone on utrophin levels in normal and DMD cultures. Key words: Utrophin, myogenesis, prednisone
dystrophin, glucocorticoids,
INTRODUCTION
Clinical trials have shown that the glucocorticoid prednisone has a long-lasting beneficial effect on muscle strength and function in Duchenne muscular dystrophy (DMD) patients [1, 2]. The molecular basis of this beneficial effect is not understood. Glucocorticoids exert a variety of effects on muscle metabolism, growth and development [3, 4]. Although certain of these effects are catabolic in nature (e.g., elevation of protease activity) positive stimulatory effects of glucocorticoids on myoblast proliferation and differentiation have been reported [5, 6]. Recent studies have examined the effect of glucocorticoids on the myogenesis of normal
~Author to whom correspondence should be addressed at: Muscle Biochemistry Laboratory, Room 665, Montreal Neurological Institute, 3801 University Street, Montreal, QC, H3A 2B4, Canada.
Duchenne muscular dystrophy,
and dystrophic muscle in vitro. The addition of ot-methylprednisolone to differentiating normal human muscle satellite cell cultures increased their content of dystrophin and myosin heavy chain [7]. This effect was attributed to inhibition of myotube death, rather than to a specific stimulation of dystrophin expression per myotube nucleus. A later study did, however, obtain some evidence indicating a specific effect of amethylprednisolone on dystrophin accumulation. Some clones established from normal muscle and most clones established from biopsies of D M D or Becker muscular dystrophy (BMD) patients, showed impaired myogenesis on exposure to o~-methylprednisolone [8]. In such clones, dystrophin accumulation was greater than in parallel, untreated, cultures [9]. Stimulation of dystrophin expression may therefore be relevant to glucocorticoid action in BMB muscle, or in those cases of D M D where some functional dystrophin is expressed. 105
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Obviously such an effect cannot explain how glucocorticoids are beneficial in cases of D M D in which no functional dystrophin is expressed. The reported effect of o~-methylprednisolone on dystrophin expression raises the possibility that glucocorticoids may also affect the expression of the dystrophin homologue, utrophin. Utrophin and dystrophin are of similar size and are homologous along their entire length, suggesting they derive from a common ancestral gene [10]. Unlike dystrophin, which is restricted in its expression to differentiated skeletal muscle, heart and brain, utrophin is expressed in a variety of fetal and adult tissues. Utrophin is, however, present at a low concentration in normal adult skeletal muscle [11], where it is restricted to the neuromuscular junction [12] and vascular components of smooth muscle [14]. In adult dystrophin-deficient muscle, utrophin is more abundant than normal and is present throughout the sarcolemma [13-15]. Assuming utrophin has a similar function to dystrophin, upregulation of utrophin expression might partially compensate for dystrophin deficiency [13, 14]. The possibility that utrophin might functionally compensate for dystrophin is supported by the recent observation that both of these proteins specifically associate with the same sarcolemmal glycoprotein complex [15]. To date the effect of glucocorticoid treatment on utrophin expression in human muscle cells has not been examined, a-Methylprednisolone has, however, been reported to enhance the accumulation of utrophin in primary cultures of mdx mouse muscle [16]. In the present study we show that dexamethasone-treated D M D muscle cultures have a higher utrophin level than control untreated D M D cultures. Interestingly, the accumulation of utrophin by normal muscle cultures was not significantly increased by dexamethasone treatment. We further show that the effect on D M D muscle cultures must be mediated post-transcriptionally, since dexamethasone has no effect on utrophin mRNA transcript levels. MATERIALS AND METHODS
Cell culture Satellite cells were obtained from biopsies of human biceps muscle taken from typical D M D
patients or, as controls, from patients with suspected neuromuscular disease, who on subsequent histological analysis had no detectable muscle pathology. The biopsies were treated with trypsin and collagenase to liberate satellite cells as previously described [17]. Satellite cells were purified by fluorescence-activated cell sorting (FACS) [18] and were grown in Ham's F12 medium supplemented with 15% fetal calf serum and Ham's serum-free supplement without dexamethasone [19]. Fresh growth medium was added every three days. At confluence the cells were switched to a medium low in serum (Dulbecco's modified Eagle's medium containing 2% horse serum) to increase differentiation. When present, dexamethasone was added at the time of the switch to DMEM, 2% horse serum. This medium was also renewed every three days until the cultures were harvested. Electrophoresis and western blotting Cultures were harvested by scraping into modified Laemmli sample buffer [20] containing 15% (w/v), SDS, preheated to 100°C and were solubilized by immediate boiling for 5 rain. Gel electrophoresis and western blotting were essentially as previously described [20, 21]. Samples corresponding to 12 Ixg of total cellular protein were analysed by SDS/PAGE on a 5.5% acrylamide, 0.75 cm thick separating gel. The gel was equilibrilated in transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3, 20% (v/v) methanol) for 15 min and electroblotted to nitrocellulose for 2 h at 100 V with cooling. Blots were rinsed with water, air dried and blocked by incubation for 40 rain in 10% skim milk in TBST (0.9% NaC1, 0.1% Tween-20, 50 mM Tris-HC1, pH 7.5). The blots were incubated for 3 h at room temperature with antibodies to dystrophin [22] or utrophin [13] at a 1 : 1000 dilution and for 1 h with second antibodies, using the ECL system (Amersham). Immunoreactive bands corresponding to dystrophin or utrophin were quantitated by video densitometry using the JAVA software program of Jandel Scientific and the values were normalized to total protein content of the cultures, determined by a modified Lowry procedure [23]. For normal cultures, utrophin and dystrophin were probed on samples from the same culture dishes. This provided an internal control for cases where dexamethasone had an effect on dystrophin accumulation, but no
Glucocorticoids and Utrophin
effect on utrophin accumulation. For analysis of the effect of different doses of dexamethasone, if the number of samples exceeded the capacity of a single blot, a set of common samples was included as an internal reference standard on each blot.
Statistical analysis of data The effect of various doses of dexamethasone on utrophin and dystrophin levels was first analysed by one way analysis of variance (ANOVA). This revealed a statistically significant effect (P<0.05) of dexamethasone on dystrophin levels in normal cells (P -- 0.003) and on utrophin levels in DMD cells (P = 0.019) but not on utrophin levels in normal cells (P = 0.8). Further analysis by ANOVA of individual group means using the Bonferroni method revealed no statistically significant differences between the effect of various doses of dexamethasone (0.1-50 ixM) on utrophin or dystrophin accumulation. Utrophin and dystrophin levels measured at all dexamethsone doses were therefore combined and compared with control values by the unpaired t-test (two tailed). This is shown in Fig. 5. The P values obtained were P<0.0001 for dystrophin in normal cells (sample number of nine for controls and 59 for dexamethasone-treated); P<0.0001 for utrophin in DMD cells (sample number of six for controls and 39 for dexamethasone-treated); P = 0.28 for utrophin in normal cells (sample number of six for controls and 38 for dexamethasone-treated).
Antibodies The antibody used to detect dystrophin was a rabbit polyclonal antibody raised against a synthetic peptide corresponding to the C-terminal 17 amino acid residues of the published human dystrophin sequence [24]. Antibodies were affinity purified before use using Affi-Gel (BioRad) conjugated to the synthetic peptide. We have previously shown this antibody does not recognize utrophin in immunoblots or in immunocytochemistry [14]. The antibody used to detect utrophin was the generous gift of Dr G. E. Morris. It is a mouse monoclonal antibody raised against a [3-galactosidase fusion protein containing 1.05 kb of sequence encoding the C-terminal domain of utrophin. The antibody has been termed MANCHO7 and has been previously shown
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not to cross-react with dystrophin in immunoblots or in immunocytochemistry [13].
Quantitation of myoblast fusion and myotube number Sister culture dishes to those used for dystrophin and utrophin analysis were rinsed with PBS, fixed with 2.5% glutaraldehyde and stained with haematoxylin and eosin. For each plate, total nuclei, nuclei in myotubes, and number of myotubes per field were counted in nine random fields at 400 × magnification. Only cells containing more than two nuclei were counted as myotubes.
Isolation of mRNA and northern blotting Polyadenylated mRNA was purified from cultured cells using the Pharmacia Quick Prep Micro mRNA purification kit. The RNA species were resolved in 1% agarose gels containing 3.7% formaldehyde. The fractionated RNA species were then blotted onto nylon membranes (Hybond-N, Amersham) by overnight capillary transfer. Blots were incubated overnight at 42°C in prehybridization buffer composed of 45% deionized formamide, 5 × SSC (20 x SSC consists of 3 M NaC1, 0.3 M sodium citrate pH 7), 500 mM sodium phosphate buffer pH 6.8, 10 x Denhardt's solution (100 × Denhardt's solution consists of 2% each of Ficoll 400, polyvinylpyrolidone, and BSA), 0.5 mg ml heat denatured salmon sperm DNA and 0.5% SDS. Blots were then incubated in hybridization buffer, containing radiolabelled cDNA probe. The hybridization buffer was composed of 50% deionized formamide, 5 × SET (20 × set consists of 3M NaCI, 0.6 M Tris-HCL pH 8, 40 mM EDTA), 1 x Denhardt's solution (as above), 0.1 mg ml1 heat denatured salmon sperm DNA and 0.5% SDS. The cDNA probes used were an 873 bp fragment (position 7002 7875) of dystrophin cDNA and a 992 bp fragment (position 6771-7703 of the EMBL sequence) of utrophin cDNA. These probes were heat denatured before addition to the hybridization buffer. Blots were incubated with the radiolabelled cDNA probe overnight at 42°C and were then washed extensively in 0.1 x SSC, 0.1% SDS buffer for 2.5 h at 65°C and subjected to autoradiography and phosphorimaging (PhosphorImager, Molecular Dynamics). Bands were quantitated using the
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ImageQuant software program (Molecular Dynamics). All mRNA levels were normalized relative to the total hybridization signal per lane obtained on probing the blot with synthetic poly U, end-labeled with [~.32p] ATP using T4 polynucleotide kinase. Hybridization conditions for poly U were as described above but at 25°C and washing was with 2 × SSC, 0.1% SDS for 1 h at 65°C. RESULTS
Effect of dexamethasone on myogenesis Both normal and D M D cultures fused extensively within 3-5 days following a switch from growth medium to serum-depleted fusion medium (Fig. 1). The initial fusion capacity of the satellite cell cultures was not affected by dexamethasone (Fig. 1, panels B and D) and was similar for normal (Fig. 1, panels A and B) and D M D (Fig. 1, panels C and D) cultures. The average % nuclei in myotubes after three days in fusion medium was 52 + 7% for normal
untreated cultures; 50 + 7% for normal dexamethasone-treated cultures; 66 + 12% for untreated D M D cultures and 61 _+ 3% for dexamethasone-treated D M D cultures. These values are close to the average final proportion of total nuclei found in myotubes of 61 + 5% reported by Blau and Webster [25] for clonal human muscle cell cultures. As originally reported by Sklar and Brown for normal cultures [7], if the cultures were subsequently maintained for a prolonged period in the absence of dexamethasone there was a gradual decline in the number of myotubes per area, starting 4-5 days after the switch to fusion medium. Table 1 shows that 15 days following the switch of D M D cells to fusion medium few myotubes remained in untreated cultures. In contrast if the cultures were maintained in fusion medium supplemented with dexamethasone, myotube survival was dramatically increased, with the optimal dose being in the range of 0.2-1.0 IxM dexamethasone. In this dose range the % nuclei in myotubes (-50%) remained close to that observed following the
Fig. 1. The effect of dexamethasone on the fusion of normal and DMD satellite cells. Satellite cells from normal human muscle (panels A and B) and from muscle of DMD patients (panels C and D) were grown to confluence and switched to fusion medium with (panels B and D) or without (panels A and C) 1 p.M dexamethasone. Cells were fixed and stained three days later. Representative fields photographed at 400 × magnification are shown. Note the presence of large multinueleate myotubes in all four panels. Quantitation of the percentage of total nuclei in myotubes in nine such randomly selected fields for each set of cells revealed no apparent effect of dexamethasone on the initial fusion of normal or DMD cultures (see text).
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Glucocorticoids and Utrophin Table 1. The effect of dexamethasone on DMD satellite cell fusion and density of myotubes Dexamethasone (~M) Experiment i 0.0 0.1 0.2 0.5 1.0 2.0 5.0 10.0 50.0 Experiment 2 0.0 0.1 0.2 0.5 1.0 5.0 10.0 50.0
Total nuclei
Nuclei in myotubes
Number of myotubes
% Nuclei in myotubes
263 294 342 228 333 268 230 233 223
+24 "+34 _+36 +_24 + 26 -+38 + 30 _+ I1 -+33
12 _+ 11 38 _+ 18 93 -+ 32 117 _+ 18 107 ± 12 67 "+20 51 +,%12 45 -+9 29 _+7
0.4 3.0 5.6 9.2 8.6 5.4 5.9 5.1 3.6
"+0.4 _+ 1.4 _+ 1.7 _+ 1.5 -+ t.0 _+ 1.3 -+ 1.2 + 1.0 +0.8
4.4 12.8 27.2 51.3 32.3 25.1 20.3 19.2 13.0
223 237 141 203 225 202 215 171
_+ 16 _+ 15 _+ 13 + 33 _+ 16 -+ 20 _+ 15 + 17
2 _+ 1 71 + 15 75 + 12 100 +_25 75 -+ 10 67 _+ 11 49 _+5 49 -+ 10
0.3 7.4 8.3 8.8 7.6 6.7 4.9 5.1
_+0.2 + 1.1 + 1.2 _+ 15. -+ 1.0 -+ 1.0 +_0.4 "+0.7
1.1 30.0 53.3 49.2 33.4 33.3 22.8 28.6
Confluent cultures were maintained for 15 days in fusion medium supplemented with the indicated concentrations of dexamethasone. The cells were fixed with glutaraldehyde and stained with haematoxylin and eosin. Nuclei and myotubes were counted in nine randomly selected fields at 400 × magnification. Values are mean _+ S.E.M. per field.
initial period of myotube formation. Similar results were obtained for normal cells (not shown).
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Effect of dexamethasone on dystrophin accumulation When normal cultures were grown and maintained in medium with or without 1 p~M dexamethasone, dystrophin levels were maintained in supplemented cultures, but declined in cultures which were continuously maintained in unsupplemented medium (Fig. 2, panel A). This confirms an earlier report of a positive effect of ot-methylprednisolone at this concentration on dystrophin accumulation by cultured human muscle cells [7]. As expected, no immunoreactive dystrophin band was detectable in D M D cultures (Fig. 2, panel B).
Utrophin accumulation by normal and DMD cultures Utrophin levels in normal cells appeared essentially unaffected by long-term dexamethasone treatment (Fig. 3, panel A). In contrast, in D M D cultures, utrophin was maintained at a higher level in dexamethasone-treated cells (Fig. 3, panel B).
Dose response We next tested the effect of various doses of dexamethasone on dystrophin and utrophin accumulation by normal and D M D cells. Such
A
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Fig. 2. The effect o f d e x a m e t h a s o n e o n the m a i n t e n a n c e o f d y s t r o p h i n levels in n o r m a l satellite cell cultures. The figure shows a w e s t e r n blot o f 12 i~g samples of t o t a l p r o t e i n f r o m n o r m a l m u s c l e cultures (panel A) a n d D M D m u s c l e cultures (panel B) m a i n t a i n e d for seven d a y s (lanes 1 & 2) a n d 15 d a y s (lanes 3 a n d 4) in fusion m e d i u m w i t h (+) or w i t h o u t (-) 1 p,M d e x a m e t h a s o n e , Blots were incub a t e d w i t h a n t i b o d i e s specific for the C - t e r m i n a l 17 a m i n o acids o f d y s t r o p h i n . T h e m a j o r i m m u n o r e a c t i v e b a n d h a d a n a p p a r e n t m o l e c u l a r w e i g h t o f - 4 0 0 k D a (arrow, p a n e l A) a n d was n o t a b l y greater in a m o u n t in d e x a m e t h a s o n e t r e a t e d cultures b y 15 d a y s after the switch to fusion m e d i u m . N o i m m u n o r e a c t i v e d y s t r o p h i n b a n d was d e t e c t a b l e in D M D cultures (panel B),
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different doses of dexamethasone used, all data obtained at the various doses were combined for further statistical analysis. Figure 5 shows that 0.2-50 p~M dexamethasone had a significant effect on utrophin accumulation by D M D cells and on dystrophin accumulation by normal cells, whereas utrophin accumulation by normal cells was not significantly affected.
Effect o f dexamethasone on m R N A transcript levels
B
To determine whether the effects of dexamethasone on utrophin accumulation may be mediated transcriptionally, m R N A was prepared from normal and D M D cultures which had been treated for either 24 h or for 5 days with dexamethasone. We deliberately used + + shorter times of treatment with dexamethasone day 7 day 15 in these experiments than were used previously Fig. 3. The effect of dexamethasone on utrophin accumula- for studies at the protein level. Our rationale tion by normal and DMD cultures. Western blot of 12 p~g was that a significant direct effect of dexasamples of total protein from normal (panel A) and DMD methasone on transcription would be detect(panel B) muscle cultures maintained for seven days (lanes 1 and 2) and 15 days (lanes 3 and 4) in fusion medium with able over this shorter period. We also wished to (+) or without (-) 1 ~M dexamethasone after reaching discriminate such an effect from indirect elevaconfluence. Blots were incubated with antibodies specific for utrophin. The major immunoreactive band had an tion of m R N A levels due to myotube rescue. apparent molecular weight of ~400 kDa and was notably Table 2 shows that neither treatment increased greater in amount in dexamethasone-treated DMD cultures the accumulation of utrophin m R N A after than in the corresponding control cultures by 15 days after the switch to fusion medium (panel B, lanes 3 and 4) but normalization for total polyadenylated m R N A present in the blot. An apparent stimulatory not in normal cultures (panel A, lanes 3 and 4). effect on dystrophin m R N A accumulation was detected. an experiment is illustrated in Fig. 4. In this experiment all cultures were harvested 15 days DISCUSSION after the switch to fusion medium, with or without added dexamethasone. Each lane represents a sample from a different culture dish. It can be seen that dystrophin levels in normal cells (panel A) and utrophin levels in D M D cells (panel C) were reproducibly higher than in controls at all doses of dexamethasone tested. In contrast utrophin levels in normal cultures were not reproducibly affected by dexamethasone treatment (panel B). The immunoreactive bands obtained at doses ranging from 0.1 to 50 ~ M dexamethasone were quantitated by video densitometry. Statistical analysis (see Materials and Methods) of the data obtained revealed a significant effect of dexamethasone on dystrophin levels in normal cultures and on utrophin levels in D M D cultures but no significant effect on utrophin levels in normal cultures. Since no statistically significant differences in utrophin or dystrophin levels were detected between the
In this study we have examined the effect of treatment o f confluent normal and D M D satellite cell cultures with dexamethasone following the switch of confluent cultures to a low-serum, fusion-promoting, medium. N o r m a l and D M D cultures responded differently. In D M D cultures a marked effect on utrophin level was observed. In contrast, in normal cultures no significant effect of dexamethasone on utrophin was detectable, although the same treatment markedly increased their dystrophin content. The effects of dexamethasone on the dystrophin content of normal cultures and the utrophin content of D M D cultures were not due to an effect on the initial stages of terminal differentiation of the cultures as they were only evident after several days of dexamethasone treatment (Figs. 2 and 3). Sklar and Brown [7] were the first to show
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7 8
9 10 11 12
A -dys
B - -utr i i~iii i~ ~ i
~ ~
~
!il
~i~ ¸
,~
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-utr
Fig. 4. The effect of various doses of dexamethasone on utrophin and dystrophin levels in normal and DMD muscle cultures. Normal (panels A and B) and DMD (panel C) muscle cultures were harvested after 15 days in fusion medium supplemented with the indicated concentrations of dexamethasone. The figure shown represents western blots of samples (12 Itg) of total protein incubated with antibodies specific for dystrophin (panel A) and utrophin (panels B and C). Arrows at the right of each panel represent the position of the major ~400 kDa immunoreactive band (dys = dystrophin; utr = utrophin). Panels A and B~lanes 1 and 2, 0 I~M dexamethasone; lanes 3 and 4, 0.2 ~,M dexamethasone; lanes 5-7, 0.5 ~M dexamethasone; lanes 8 and 9, 1.0 p,M dexamethasone; lanes 10-12, 2.0 p,M dexamethasone. Panel C--lanes 1-4, 0 p.M dexamethasone; lanes 5-8, 0.1 IxM dexamethasone; lanes 9-11, 0.2 txM dexamethasone; lane 12, 0.5 ~M dexamethasone.
that glucocorticoids can increase the dystrophin content of human satellite cell cultures by inhibiting the naturally occurring death of mature myotubes. In the present study we show a similar long-term effect of dexamethasone on utrophin accumulation by DMD muscle cultures. This effect was associated with a greater number of myotubes present in DMD cultures maintained long-term in dexamethasone-supplemented fusion medium. Taken together these findings suggest that rescue of myotubes by glucocorticoid treatment may also be a significant factor in the observed stimulatory effect of dexamethasone on utrophin accumulation by DMD satellite cell cultures. It should be noted, however, that the correlation with myotube rescue was not perfect. Dexamethasone had a marked biphasic dose-depen-
dent effect on myotube survival (Table 1) over the range 0.1-50 IxM, whereas no significant differences were noted between different doses over this range for the dexamethasone effect on utrophin or dystrophin accumulation. Dystrophin is a muscle-specific protein which is known to increase during myoblast differentiation [26], it can therefore readily be seen how rescue of mature myotubes from cell death could cause a relative increase in dystrophin accumulation. The mechanism by which myotube rescue could increase the relative content of utrophin is less obvious. Utrophin is not muscle specific and does not increase in muscle cultures as a consequence of differentiation [14]. Passaquin et al. [16] have recently reported that the glucocorticoid ot-methylprednisolone
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Utrophin (Duchenne)
Fig. 5. Quantitative analysis of the effect of dexamethasone on utrophin and dystrophin levels in normal and D M D satellite cell cultures. Normal and D M D satellite cells were maintained for 15 days in fusion medium with (hatched bars) or without (open bars) 0.1-50 ~M dexamethasone. Cells were harvested and 12 ~g samples were immunoblotted as described for Fig. 4. The ~400 kDa immunoreactive bands were quantitated by video densitometry. The values shown represent mean + S.E,M. Further details of the statistical analysis are given in the Materials and Methods section. The asterisks above the hatched bars indicate a statistically significant increase in dystrophin or utrophin levels, compared to the untreated controls.
increases the utrophin content of m d x mouse muscle cultures. A similar protocol was used to that in the present study, in that the glucocorticold was added at the onset of myoblast fusion. In the m d x study an effect on utrophin accumulation was evident after seven days of a-methylprednisolone treatment and this correlated with a stimulatory effect on myotube numbers. The antibody used to detect utrophin in the m d x studies cross-reacted with dystrophin, making it impossible to tell whether c~methylprednisolone also stimulated utrophin accumulation in normal mouse muscle.
Collectively, the results of this study and the study of Passaquin et al. [16] clearly show that glucocorticoid treatment of dystrophindeficient human and mouse muscle cultures, respectively, can enhance their content of utrophin relative to other cellular proteins. The question remains whether this is due to stimulation of transcription, or increased stability of mRNA or protein. Two other findings in the present study provide further insight into the mechanism underlying this effect. First is the observation that there was no detectable effect of dexamethasone on utrophin mRNA transcript levels. This suggests that the effects of dexamethasone on utrophin accumulation are mediated post-transcriptionally. Interestingly, we did obtain some indication of a stimulatory effect of dexamethasone on dystrophin mRNA accumulation. Further studies are required to demonstrate the reproducibility of this effect. A positive effect of dexamethasone on dystrophin mRNA levels would support recent studies at the protein level [9,16] which suggest that glucocorticoids may selectively effect dystrophin expression in cultured muscle cells. As discussed by Hardiman et al. [9] the muscle promoter for dystrophin does not contain a glucocorticoid response element [27] indicating that a transcriptional effect of dexamethasone on dystrophin expression would have to be indirect or be mediated via an alternate promoter, such as the brain promoter. It has not yet been established whether any of the dystrophin promoters have a glucocorticoid response element. Secondly, we observed that the effect of dexamethasone on utrophin accumulation was
Table 2. The effect of dexamethasone on utrophin and dystrophin mRNA levels in normal and DMD satellite cell cultures Dexamethasone treatment Normal cells DMD cells
None 24 hr 5 days None 24 hr 5 days
Dystrophin/poly A
Utrophin/poly A
1.0 1.4 1.7 n.d. n.d. n.d.
1.0 0.8 1.1 1.0 0.9 1.1
Confluent cultures were grown under three different conditions: (i) for five days in fusion medium without added dexamethasone; (ii) for four days in fusion medium without dexamethasone followed by 24 h in medium supplemented with 1 IxM dexamethasone; (iii) for five days in fusion medium supplemented with l IxM dexamethason. Cultures were harvested and mRNA was isolated and analysed by northern blotting. Values shown are the mean of two separate experiments and are normalized to poly (A) mRNA, with the control values arbitrarily set at 1.0. Note that normalization to measured poly (A) levels indicates dexamethasone did not selectively alter utrophin mRNA levels.
Glucocorticoids and Utrophin
much more dramatic in DMD cultures than it was in normal cultures. Any mechanism proposed to explain the effect of dexamethasone on utrophin accumulation by D M D cells must take into account this relative lack of effect of dexamethasone on utrophin accumulation by normal cells. Furthermore, regardless of the explanation of this observation, it clearly indicates that the effect of dexamethasone on utrophin accumulation in D M D cells is largely a secondary consequence of dystrophin deficiency. One possible mechanism is suggested by the recent observation that utrophin and dystrophin bind to the same sarcolemmal glycoprotein complex [15]. Given this fact, it is tempting to speculate that utrophin may be more stable in D M D myotubes than in normal dystrophin-containing myotubes, because in D M D cells utrophin does not have to compete with dystrophin for binding to the dystroglycan complex. According to this model, utrophin bound to the dystroglycan complex would be less susceptible than unbound utrophin to protein turnover and would therefore accumulate to a greater extent within the myotube. Dexamethasone, by rescuing mature myotubes from cell death, would therefore increase the relative utrophin content of DMD cultures over that of untreated cultures. This model of increased accumulation of utrophin in D M D myotubes compared to normal myotubes as a secondary consequence of decreased competition with dystrophin is also consistent with the observation of increased accumulation of utrophin in D M D and BMD muscle [14, 28]. Marked up-regulation of utrophin in extraocular muscle and cardiac muscle of the mdx mouse has been shown to be associated with a corresponding increase in the 50 and 156 kDa components of the dystrophin/glycoprotein complex [15] providing further evidence of an interaction between utrophin and the dystroglycan complex in vivo. A further factor which could contribute to the dexamethasone effect on utrophin, is that a large protein like utrophin may be more stable in a mature non-dividing cell like a myotube than it is in a dividing satellite cell, in which the membrane cytoskeleton is constantly being disassembled and reassembled during cell division. While such an effect would clearly favor utrophin accumulation in both normal and D M D cells, competition with dystrophin for
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membrane binding sites may counteract this benefit and lower the stability of utrophin in normal myotubes. In conclusion, although glucocorticoids may be beneficial in D M D by aiding the regeneration process (e.g., by increasing the survival of newly regenerant myotubes prior to innervation) and consequently stabilizing utrophin levels in mature myotubes, the present study indicates that they do not act directly to increase utrophin expression. are grateful to the Muscular Dystrophy Association of Canada, the Muscular Dystrophy Group of Great Britain, the Muscular Dystrophy Association (U.S.A.) and the Medical Research Council for financial support. F. P. was the recipient of a University of Toronto Medical Alumni Fellowship. We also thank Dr G. Morris for the MANCHO7 antibody and Dr J. Tinsley for assistance in isolation of the original cDNA clones used in this study. Acknowledgements--We
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