Multiple effects of interferon on myogenesis in chicken myoblast cultures

Biochimica et Biophysica A cta, 804 (1984) 370- 376 Elsevier

370

BBA 11322

MULTIPLE EFFECTS OF INTERFERON ON MYOGENESIS IN CHICKEN MYOBLAST CULTURES Y O S H I M I T O M I T A a and S H U J I H A S E G A W A

b

Department of Microbiology and I, Brain Research Institute, School of Medicine, Chiba University, Chiba 280 (Japan) (Received November 23rd, 1983)

Key words: Interferon; Myogenesis," (Chicken myoblast)

Effects of chicken interferon on the differentiation of chicken skeletal muscle in vitro were examined. Continuous treatment of chicken myoblast culture with 200 I U / m l of interferon (10 I U / m g protein) resulted in significant inhibition of cell fusion and subsequent myotuhe formation. However, treatment of myoblast culture with 2 to 200 I U / m l of interferon increased activities of creatine kinase and myokinase in 4- or 6-day cultured muscle cells in a dose-dependent fashion. The effect of interferon on myokinase was less than on creatine kinase. Three-fold increase in creatine kinase activity induced by interferon was not accompanied by the accelerated transition of creatine kinase isozyme from BB- to MM-type. On the other hand, accumulation of acetyicholinesterase in interferon-treated cells at day 6 was suppressed to nearly half the level of control cells. Rates of actin and myosin synthesis in 4-day cultures estimated by pulse-labelling with [3SS]methionine were also suppressed to 85% of control cultures. However, a proportion of 3sS-laheiled actin and myosin in labelled proteins associated with glycerinated cells was not changed by interferon treatment. These results indicate that partially purified interferon has multiple effects on the process of the myogenic differentiation of chicken myoblast in vitro.

Introduction Interferon exerts various non-antiviral effects on cells, such as the reduction of growth rate [1] and the suppression of virus-induced fusion of cells [2-5] by inducing alterations in plasma membranes, cytoskeletal structures and metabolic activities [6-11]. Interferon is also known to modulate cell differentiation in vitro [12-15]. Myogenesis in vitro seems to be suitable for the study of non-antiviral actions of interferon on cell fusion and differentiation. Myoblasts from chicken and mammalian embryos proliferate, differentiate and fuse in culture, to form multinucleated myotubes. During the myogenic process the amount of contractile proteins and enzymic activities of creatine kinase Abbreviation: Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid. 0167-4889/84/$03.00 © 1984 Elsevier Science Publishers B.V.

(EC 2.7.3.2) myokinase (EC 2.7.4.3) and acetylcholinesterase (EC 3.1.1.7) rapidly accumulate in cells to a high level [16-19]. Recently, Lough et al. [20] reported inhibitory effects of chicken interferon on myogenic events during the differentiation of skeletal muscle in vitro. In this paper we report that chicken interferon has multiple effects on the differentiation of cultured chicken myoblasts. Namely, interferon enhances the accumulation of creatine kinase and myokinase in muscle cells in a condition in which the fusion of myoblasts and the accumulation of acetylcholinesterase are markedly inhibited. Materials and Methods Cell cultures. Muscle cell cultures were prepared from thigh muscles of 11- or 12-day-old chick embryos, as described previously [21]. Briefly,

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ferences between control and interferon-treated cultures were observed by day 4 after the beginning of interferon treatment. Many long and thick myotubes having cluster of nuclei at their peripheral or branching regions developed in control cultures (Fig. lc), but short and thin myotubes were formed in interferon-treated cultures (Fig. ld). At day 6, myotubes in control cultures became longer and wider, whereas the development of myotubes in interferon-treated cultures remained inhibited (Fig. le and f). Fig. 2a shows that myoblast fusion was initiated at 48 h and completed by 80 h after plating in high-density cultures. The addition of interferon (100 IU/ml) to cultures resulted in a reduction of their fusion

indexes to about 40~ of those of control cultures at 90-117 h after plating. However, when the interferon-treatment started after the onset of fusion (at day 3), subsequent fusion was not inhibited significantly (Fig. 2b), suggesting that the anti-fusion state is efficiently induced in myoblasts prior to fusion. To confirm that diminished formation of myotubes can be attributed to an action of interferon, we examined responsiveness of muscle culture to interferon by estimating 2-5A synthetase activity induced by interferon. Myoblast cultures were treated with interferon from 48 to 72 h after the time of plating. Synthesis of radioactive 2-5A in extracts of cells treated with 10 and 100 I U / m l of interferon was 190 and 2909 cpm/80 #1 per 25

\

Fig. 1. Inhibition of myotube formation by interferon-treatment. Myoblasts were cultured for 2 days (a, b) or 4 days (c, d) in the absence (a, c) or presence (b, d) of 100 I U / m i of interferon, and photographs were taken under phase-contrast microscope ( × 62.5). Myotubes formed after 6-days cultivation in a control dish (e) and in a dish treated with 200 I U / m l of interferon (f) ( × 125).

371 (1.5-2.2). 107 cells were seeded on a gelatin-coated 150 mm petri dish and maintained in Eagle's minimal essential medium, containing 15% horse serum and 5% chick embryo extract, for 16 to 18 h. Secondary cell suspension was prepared from primary cultures to eliminate fibroblasts and to enrich myogenic cells in culture. About 3.2-105 cells were inoculated on a gelatin-coated 35 mm dish, unless otherwise stated, and were grown in the nutrient medium described above. Cultures were fed fresh nutrient medium every 2 days. When cultures were treated with interferon from day 1, interferon was added 6 h after plating. Interferon. Partially purified chicken interferon, which was induced by ultraviolet-inactivated Newcastle disease virus in chicken embryo fibroblasts in primary culture, was kindly supplied by Dr. M. Kohase, National Institute of Health, Japan. The specific activity of interferon was 1.0.106 I U / m g protein. Fusion index. Duplicate cultures treated with or without interferon were fixed with 100% methanol for 10 min and stained with 10% Giemsa solution in phosphate-buffered saline (pH 6.8) for 20 min. Nuclei were counted under a microscope with an eyepiece grid. For each dish, 20 randomly selected microscopic fields were examined, and in total 1600-2500 nuclei were counted. The extent of cell fusion is expressed as a fusion index, i.e., the percentage of nuclei found in multinucleated cells. Assay of enzyme activities and contents of DNA and protein. Cells were scraped from dishes with a rubber policeman, washed by suspending in Eagle's minimal essential medium and dispersed in 0.5 ml of 20 mM mM Tris-HC1 buffer (pH 8.0). The cells were disrupted by brief sonication. Creatine kinase activity and myokinase activity were measured by the method described by Kuby et al. [22] and Shainberg et al. [18], respectively. For determination of activity of acetylcholinesterase, Triton X100 was added to cell homogenates to a concentration of 0.1%. The enzyme activity was measured in the presence of 0.1 mM tetraisopropylpyrophosphoramide, according to the procedure described by Ellman et al. [23]. One unit of enzyme activity is the amount which catalyzes the formation of 1 /xmol product/min at 30°C for creatine kinase and at 37°C for the other two enzymes. 2-5A synthetase activity was estimated as described pre-

viously [4]. For 2-5A synthesis assay, cells detached from three dishes were sonicated in 0.15 ml of 20 mM Hepes-KOH buffer (pH 7.4)/60 mM magnesium acetate/0.2 M KCI, and 80 /~1 of the sonicate was used for enzyme assay. DNA content of cell homogenates was measured by indole reaction [24], and protein content was determined by the method of Lowry et al. [25]. Zone electrophoresis. Electrophoresis was performed in 0.05 M barbital/0.01 M 2-mercaptoethanol (pH 8.6) on cellulose acetate strips at 0.6 m A / c m for 120 min. The strips were stained for creatine kinase activity as described previously [26]. Actin and myosin synthesis. Muscle cultures were washed with Eagle's minimal essential medium and labelled for 90 min with [35S]methionine (20 /~Ci/ml; Amersham) in minimal essential medium with 200-fold reduction in the methionine concentration. The cultures were rinsed with minimal essential medium and kept in 50% glycerol/50% standard salt solution at 4 ° C for 4 days [27]. Standard salt solution consisted of 0.1 M KC1/5 mM MgCI2/6 mM sodium phosphate buffer (pH 7.0). The glycerol solution was removed, and the preparations were rinsed with standard salt solution, scraped into standard salt solution and precipitated by centrifugation at 3000 rpm for 5 min. The pellets were solubilized with 100/~1 of sample buffer for electrophoresis, 0.15 M Tris-HC1 (pH 6.8)/30% sodium dodecyl sulphate (SDS)/3% 2mercaptoethanol/30% glycerol, and were applied on a gel for electrophoresis. Slab gel electrophoresis was done in gel of 12.5% acrylamide containing 0.1% SDS. Bands of actin (molecular weight 43 000) and myosin heavy chain (molecular weight 200 000) were located by fluorography [28]. Radioactivities in these b~nds were determined by cutting the dried gel and counting the activities in ACS II scintillant (Amersham). R ~

Effects of interferon on myotube formation Myoblast fusion and morphological changes during myogenic differentiation in control and interferon-treated cultures were studied by determining fusion indexes and by microscopic observation. As shown in Fig. 1, morphological dif-

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respectively, whereas synthesis of 2-5A in control extract was hardly detectable.

creatine kinase and myokinase activities in myotube cultures. Interferon treatment at 200 I U / m l for 4 days from 48 h after plating resulted in three fold increase of the specific activity of creatine kinase; creatine kinase activities of interferon-treated cultures and control cultures were 72.4 _ 1.9 and 23.8 _+2.4 m U / m g protein (mean _+ S.D. for three independent experiments), respectively. As shown in Table I, the effect of interferon on creatine kinase activity was dose-dependent. A 1.5-fold enhancement of creatine kinase level in cultured muscle cells was observed even after 2-days treatment from day 2 to day 4 with 100 I U / m l of interferon. But 1-day treatment from day 5 to day 6 was not enough to ensure the significant enhancement of creatine kinase level (data not shown), suggesting that interferon accelerates creatine kinase accumulation at a relatively early stage of myotube formation in vitro. Myokinase activity was also stimulated by interferon treatment, but less effectively as compared to creatine kinase activity. Enzyme activity of myokinase in interferon-treated cultures (100 IU/ml) was 122.5% of control (Table I).

Enhanced accumulation of creatine kinase and myokinase in interferon-treated cells Interferon stimulated the accumulation of both

Creatine kinase isozymes To determine whether the enhanced accumulation of creatine kinase activity by interferon was

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Fig. 2. Myoblast fusion in interferon-treated and untreated cultures. Interferon (100 I U / m l ) was added to cultured 6 h (a) or 3 days (b) after plating and culture medium with or without interferon was changed at 2-day intervals. Inoculum size was 3.2.105 cells (a) and 6.4.104 cells (b) per dish. O and zx, untreated control cultures; • and =, interferon-treated cultures. Triangle symbols represent a separate experiment. A260,

TABLE I D O S E - D E P E N D E N T EFFECTS OF I N T E R F E R O N T R E A T M E N T ON E N Z Y M E ACTIVITIES IN C U L T U R E D M Y O T U B E S Myotube cultures obtained from a single muscle cell preparation were treated with interferon (IFN) for 4 days from 48 h after plating, harvested and subjected to simultaneous analysis of enzyme activities and D N A and protein content in Expt. 1. Myotube cultures obtained from another muscle cell preparation were treated with interferon for 6 days from day 1, harvested and subjected to measurement of myokinase in Expt. 2. IFN (IU/ml)

Expt. 1 0 2 20 200 Expt. 2 0 10 100

DNA ( # g / d i s h a)

7.38±0.94 7.70±1.15 6.51±1.01 6.91±0.74

Protein ( # g / d i s h a)

223± 6.6 256±13.9 2 ~ ± 1.4 235±16.0 345 + 14.1 315 ± 4.1 411 ± 2.8

Creatine kinase mU/mg %

Acetylcholinesterase mU/mg %

Myokinase mU/mg

protein a

protein b

protein a

27.1±4.4 42.8±7.7 50.5±5.4 73.1±6.2

1~ 157 186 270

32.6 25.0 24.6 17.7

%

1~.0 76.7 75.5 54.3 80.0±6.9 87.5±3.3 98.8±8.7

" Each value is the mean + S.D. for three dishes. b Aliquots of homogenates obtained from three dishes were pooled for determination of enzyme activity.

100.0 109.4 122.5

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acetylcholinesterase accumulation in cultured muscle cells. As shown in Table I, in contrast to the effects on creatine kinase and myokinase, interferon treatment suppressed acetylcholinesterase accumulation in a dose-dependent fashion. Acetylcholinesterase activity in cultured cells which were treated with 200 I U / m l of interferon for 4 days before harvesting was 54.3% of that in control cultures. IFN

3

Effect of interferon on actin and myosin synthesis

4-

rBB

BMMM

--

Fig. 3. Electrophoretic patterns of creatine kinase isozymes. Extracts were prepared from 6-day cultures of muscle cells grown in the presence or absence of interferon (200 IU/ml). C, control; IFN, interferon-treated. accompanied by an accelerated transition in creatine kinase isozyme from BB-type to MM-type, we examined the isozyme patterns of creatine kinase in cell homogenates prepared from interferontreated and untreated cultures on day 6. As shown in Fig. 3, both preparations contained BB-, BMand MM-type creatine kinases, and the electrophoretic pattern of the interferon-treated preparation was extremely similar to that of control, suggesting that interferon equally stimulated the accumulation of both B- and M-type subunits of creatine kinase.

Suppression of acetylcholinesterase accumulation in interferon-treated cells We examined the effect of interferon on

Muscle cells were grown in the presence or absence of interferon (200 I U / m l ) and the rates of actin and myosin heavy chain synthesis were determined on day 4 by pulse labelling of cells with [35S]methionine followed by glycerination and analysis with SDS-polyacrylamide gel electrophoresis. Table II shows that, although total protein content in interferon-treated culture was higher than that in control culture (121.3%), an incorporation of [35S]methionine into trichloroacetic acidinsoluble materials of glycerinated cells in interferon-treated culture was lower than that in control culture (86.9%). An almost similar gel-electrophoretic pattern was observed in both samples (Fig. 4). Radioactivities incorporated into actin and myosin heavy chain bands in interferon-treated culture were also lower than those in control culture (86.8 and 82.2% of control, respectively). However, the sum of the radioactivities recovered in actin and myosin bands was 57.2% of trichloroacetic acid-insoluble radioactive materials applied on gel in the control preparation and 56.3% in the interferon-treated preparation. These results sug-

TABLE II EFFECT OF INTERFERON-TREATMENT ON THE INCORPORATION OF [ 35S]METHIONINE INTO ACTIN A N D MYOSIN IN C U L T U R E D MUSCLE CELLS Muscle cells were grown with or without interferon (IFN), pulse-labelled with [35S]methionine, glycerinated and solubilized. Radioactivities of myosin and actin bands in gels shown in Fig. 3 were determined after cutting the gels. TCA, triehioroaeetic acid. TCA-insoluble radioactivity in glycerinated cells

Control IFN-treated

Radioactivity recovered in myosin

in actin

cpm/dish

% control

cpm

% recoverya

epm

% recovery a

114 370 99 340

100.0 86.9

4613 4003

40.3 40.3

1 937 1 592

16.9 16.0

a Percentage of radioactivity applied on the gel.

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Mr.

94, 67, 43, 36,

4

Fig. 4. SDS-polyacrylamide gel electrophoresis of [35S]methionine-labelled materials in glycerinated muscle cells. Myoblasts were grown in the absence or presence of interferon (200 I U / m l ) for 4 days. For analysis, radioactive materials from control and interferon-treated cells (11437 and 9930 trichloroacetic acid-precipitable cpm, respectively) were applied on the gel. Eiectrophoresis was carried out at 160 V for 3.5 h. C, control; IFN, interferon-treated; A, actin; M, myosin-heavy chain. M r ( × 10-3): 220, ferritin half unit; 94, phosphorylase b; 67, albumin; 43, ovalbumin; 36, lactate dehydrogenase.

gest that interferon decreased the rate of synthesis of proteins associated with glycerinated cells by nearly 15% in 4-day cultures of muscle cells, but did not change the proportion of synthesis of actin and myosin in those proteins. Similar results were obtained in experiments using 6-day cultures (data not shown). Discussion

Recently we have shown that interferon inhibits cell fusion of human transformed cells which are induced by ultraviolet-inactivated retrovirus [2] and Sendai virus [3,4]. This action of interferon was further confirmed by Chatterjee et al. [5] using human and monkey cells. In the studies reported here, interferon at concentrations up to 200 I U / m l inhibited cell fusion in chick embryonic myoblast cultures and affected the accumulation of some muscle-specific proteins in cultured cells. Interferon increases the rigidity of plasma membrane lipid bilayers in mouse sarcoma S-180 cells [10] and the reorganization of actin-containing microfilaments in cells, which may result in the suppression of redistribution of some cell surface components [9]. It is likely that the similar alterations in

plasma membrane and submembraneous cytoskeletal components are induced in interferontreated myoblasts. The inhibition of myoblast fusion by interferon treatment could be a result of an antagonistic effect of interferon on the rapid increase in the membrane fluidity in myoblast, which precedes the membrane union [29]. Myoblast fusion is a multistep process, including at a minimum two phases: (i) cell migration, recognition and alignment and (ii) membrane union [30]. An alternative explanation of fusion-blocking action of interferon is that the interferon-induced suppression of synthesis or redistribution of cell surface components which are required for cell migration, recognition and alignment might result in blockade of the fusion process. Originally, Lough et al. [20] tested effects of chicken interferon on the chicken myogenesis in vitro and reported that interferon inhibits myogenesis in terms of morphology and isozyme transition from BB-creatine kinase to MM-creatine kinase. In our present work, we confirmed the inhibitory action of interferon on myotube formation under light-microscopic observation. However, we found that, up to 200 I U / m l , chicken interferon enhances the accumulation of creatine kinase and myokinase in muscle cells, even in a condition in which interferon inhibits the fusion of myoblasts, the accumulation of acetylcholinesterase and the synthesis of contractile proteins. We also found that the transition of BB-creatine kinase to MM-creatine kinase is not inhibited by interferon treatment. It may be argued that the diminished formation of myotubes and the alterations in enzyme activities are due to an action of some components other than interferon, which were produced by chicken embryonic fibroblast cells and coexisted in our interferon preparation. This is unlikely, since myoblasts were grown in nutrient medium supplemented with the supernatant of homogenate of whole chick embryos. Our observations that in primary muscle cultures the enzyme activity of creatine kinase and myokinase was elaborated in fusion-blocked myoblast cells agree with the findings reported in non-fusing muscle cell line M3A [31] that the synthesis of creatine kinase and the progress of cell fusion are regulated independently in myogenesis in vitro.

376 W h i l e o u r e x p e r i m e n t s were in progress, Fisher et al. [32] r e p o r t e d an acceleration of m y o b l a s t fusion and creatine kinase isozyme transition by h u m a n leukocyte interferon in h u m a n m y o b l a s t culture. In chicken m y o b l a s t culture, we observed a slight acceleration in m y o b l a s t a l i g n m e n t in early stage of cultures w h ic h were treated with relatively high titre of interferon (1000 I U / m l ) , even t h o u g h following fusion was m a r k e d l y inhibited. L o n g t e r m t r e a t m e n t with 1000 I U / m l of i n t e r f e r o n was toxic for culture, a n d m a n y u n f u s e d cells were d e t a c h e d f r o m a dish by days 5 to 6 after the b e g i n n i n g of treatment. At present, we c a n n o t p r o p o s e a reason for the d i s c r e p a n c y observed on the effects of i n t e r f e r o n on m y o b l a s t fusion in h u m a n and chicken cultures. In o u r system, it is c o n c e i v a b l e that i n t e r f e r o n has divergent effects on m y o g e n i c process of chicken skeletal muscle in vitro. A c k n o wl ed g m en t s W e wish to t h a n k Dr. M. K o h a s e for kindly suppling chicken interferon. This study was supp o r t e d by grants f r o m N a t i o n a l C e n t e r for N e r v o u s , M e n t a l a n d M u s c u l a r D is o r d e r s a n d the research c o m m i t t e e of C N S d e g e n e r a t i v e diseases o f the M i n i s t r y of H e a l t h and Welfare, J a p a n a nd a g r an t f r o m the M i n i s t r y of E d u c a t i o n , Science a n d Culture, Japan. 1 Stewart, W.E. 11 (1981) The Interferon System, SpringerVerlag, Wien 2 Tomita, Y. and Kuwata T. (1979) J. Gen. Virol. 43, 111-117 3 Tomita, Y. and Kuwata T. (1981) J. Gen. Virol. 55,289-295 4 Tomita, Y. Nishimaki, J., Takahashi, F. and Kuwata T. (1982) Virology 120, 258-263 5 Chatterjee, S., Cheung, H. and Hunter, E. (1982) Proc. Natl. Acad. SCi. USA 79, 835-839 6 Friedman, M.R. (1979) in Interferons (Gresser, I., ed.), Vol. 1, pp. 53-74, Academic Press, New York 7 Pfeffer, L.M. Wang, E. and Tamm, I. (1980) J. Cell Biol. 85, 9-17

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