Apolipoprotein A-l Expression Is Resistant to Dimethyl Sulfoxide Inhibition of Myogenic Differentiation DAVID LOURIM AND JIM JUNG-CHING LIN’ Department
trophysiological differentiation . In contrast, DMSO reversibly inhibited myogenic differentiation [2,7-g], as well as aspects of chicken chondrogenic differentiation [lo]. The mechanisms by which DMSO induces these opposing responses in diverse cell types are not understood but are suggested to involve transcriptional regulatory events [8, 111. We have recently reported that treatment of chick embryo myogenic (CEM) cells with DMSO resulted in the reversible inhibition of myofibrillar protein and nuclear lamin A expression . We also observed an unidentified protein of approximately 26 kDa whose synthesis appeared to be resistant to or induced by DMSO treatment. In the present study, based on cellular, biochemical, and immunological analysis, this protein has been identified as apolipoprotein A-l (APO A-l). Apo A-l is the major protein component of high density lipoproteins (HDL’s) (for reviews, see [12-141) and serves as an activator of lecithin:cholesterol-acyltransferase [ 151.In the adult chicken, the liver and intestine are the major sites of Apo A-l synthesis [12, 161. However, 1 week preceeding and following hatching, skeletal and cardiac muscle were found to synthesize Apo A-l at approximately 34 times the rate observed in the adult skeletal and cardiac tissues [17-191. Apo A-l synthesis by developing skeletal muscle of the peripheral tissues is suggested to function in a process known as “reverse cholesterol transport” [ 16, 201. The differential expression of Apo A-l in chick developing skeletal muscle is transcriptionally regulated [ 171 and has been suggested to be tightly linked to muscle differentiation . We report here that Apo A-l is expressed in a manner temporally similar to muscle-specific proteins in cultured CEM cells, and due to its secretion into the culture medium, Apo A-l has a half-life in cell homogenates of 23 min. Despite the DMSO-induced inhibition of musclespecific protein expression, Apo A-l expression and secretion remained unaffected. These results suggest that expression of the Apo A-l and muscle-specific sarcomerit genes is regulated by independent mechanisms.
Primary cultures of chick embryonic muscle (CEM) were analyzed for the differential expression of a 26kDa protein during myogenesis. We have identified this 26-kDa protein as apolipoprotein A-l (Apo A-l), the major protein of serum high density lipoprotein particles. Apo A-l was expressed in a pattern temporally similar to those of muscle-specific proteins, by myoblasts at very low levels, and by myotubes at high levels. The half-life of Apo A-l in CEM cell homogenates was 23 min. This fast turnover rate appeared to be due to the secretion of Apo A-l into the culture medium. To further characterize the relationship of Apo A-l expression and myogenic differentiation, CEM cultures were treated with dimethyl sulfoxide (DMSO). In the presence of 2% DMSO, myotubes exhibited an atrophied morphology and an inhibition of the synthesis and accumulation of muscle-specific sarcomeric proteins. During recovery from DMSO treatment, the expression and accumulation of muscle-specific proteins returned to high levels. In contrast, the rates of synthesis and secretion of Apo A-l in control, DMSO-treated, and DMSOrecovered CEM cells were nearly equivalent. These results indicate that the expression of Apo A-l is not strictly linked to the expression of muscle-specific sarcomeric proteins in skeletal muscle and suggest that independent, or additional regulatory mechanisms exist which modulate Apo A-l expression during myogenesis. 0 1991 Academic Press, Inc.
INTRODUCTION Myogenesis is characterized by the conversion of replicating, mononucleated myoblasts to syncytial myotubes which express a battery of unlinked muscle-specific genes. A number of chemical and biological agents have been useful in investigating potential mechanisms involved in regulating the expression of muscle-specific genes [l, 21. Dimethyl sulfoxide (DMSO) is capable of stimulating leukemic cells to undergo differentiation through several distinct pathways [3-51 and stimulating neuroblastoma cells to undergo morphological and elec1 To whom reprint
of Zowa, Iowa City, Iowa 52242
MATERIALS AND METHODS Cell culture. Chick embryo myogenic cells were isolated from leg muscle of lo-day-old embryos by a modification  of the procedure
requests should be addressed. 57
Copyright 0 1991 hy Academic Press, Inc. All rights of reproduction in any form reserved.
of Konigsberg . Cells were maintained in Dulbecco’s modified Eagles (DME) medium containing 15% horse serum and 2% chick embryo extract (complete medium) and incubated at 37°C in a hum&fied chamber with 5% CO, and 95% air. Chick embryo fibroblasts (CEF) were prepared as described  and maintained in DME medium containing 10% fetal calf serum (FCS). For DMSO treatment, CEM cells were cultured for 12 to 24 h before refeeding the cells with complete medium containing 2% DMSO. Control cultures were refed at the time of DMSO addition. For the DMSO-reversal experiments, CEM cells were cultured in complete medium plus DMSO for the indicated times, then washed and refed with complete medium, and incubated for the indicated additional period of time, For immunofluorescence experiments, cells were grown on 12-mm round, collagencoated glass coverslips. For scanning electron microscopic analysis, CEM cells were fixed in 2% glutaraldehyde (in 0.1% cacodylate buffer, pH 7.1) then subjected to ethanol dehydration followed by critical point drying and platinum shadowing. Samples were photographed on a JEOL scanning electron microscope at an accelerating voltage of 5.0 kV. Antibodies. The anti-tropomyosin monoclonal antibody CHl which recognizes the skeletal muscle a- and @-tropomyosin isoforms was prepared and characterized as previously described . Anti-Atype lamin antibody (C23) was prepared and characterized as described . The rabbit anti-chicken apolipoprotein A-l antiserum was the generous gift of H. Lebherz (San Diego State University, San Diego, CA). The rabbit anti-p26 antiserum (rabbit 7) was prepared as previously described . Gel electrophoresis and immunoblotting. Cultured cells, after washing three times with phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 1.5 m&f KH,PO,, 8.0 mM NasHPO,, pH 7.3) containing 5 mM MgCl, and 0.2 mM EGTA, were solubilized by the addition of sample buffer containing 0.1 M dithiothreitol, 2% SDS, 80 m&f Tris, pH 6.8, and 15% glycerol. The samples were placed into a boiling water bath for 3-5 min, passed 5-10 times through a 27-gauge syringe needle, and then returned to the water bath for an additional 2-3 min before centrifugation and storage of the supernatant at -20°C. Total cellular proteins from control and DMSO-treated CEM were analyzed at various times after plating by gel electrophoresis and immunoblot analysis. SDS-PAGE was carried out according to Laemmli  with a low concentration of bisacrylamide (12.5% acrylamide and 0.104% bisacrylamide). Two dimensional (2D) gel electrophoresis was performed according to the method of O’Farrell , using 4% pH 4-6 or pH 5-7 ampholines and 1% pH 3.5-10 ampholines in the first dimension gel mixture. Protein immunoblotting was performed according to Towbin et al. , as modified by Lin et al. . For &orography and autoradiography of gels, a modified method of Bonner and Laskey  was performed as previously described . RNA isolation and in vitro translation. Total RNA and poly(A)+ RNA were isolated from the indicated cultured cell types by the guanidinium isothiocyanate method and oligo(dT) cellulose column chromatography as described by Maniatis et al. . In vitro translation reactions were carried out in rabbit reticulocyte lysates (Promega, Madison, WI), in the presence of 10 &i/ml [!?S]methionine (1200 Ci/mmol), with either 10 c(g of total RNA or 1 pg of poly(A)+ RNA. TO stop the reactions, an equal volume of sample buffer was added to each reaction tube. Samples were then processed for gel analysis and immunoprecipitation as for cell homogenate samples. Radioactive labeling and immunoprecipitation. For long-term labeling experiments, CEM cells were grown in 35-mm culture dishes and incubated for 5-12 h in the presence of 20-50 &i/ml of [s’S]methionine (1200 Ci/mmol) in 1 ml of methionine-free medium containing 2.5% FCS. For pulse-chase experiments, cells grown in 35-mm dishes were incubated with methionine-free medium containing 2.5% FCS for 30 min before brief pulses (4-30 min) with 20-100 &i/ml of [35S]methionine in methionine-free DME medium contain-
ing 2.5% FCS. Chases were initiated by adding cold methionine to a final concentration of 5 n&f. Total trichloroacetic acid precipitable counts in total cell homogenates did not increase with chase time. Culture media was exhaustively dialyzed in 10 mit4 Tris, pH 7.5, 1 mM phenylmethylsulfonyl fluoride at 4”C, and then lyophilized and resuspended in sample buffer. Immunoprecipitation procedures were carried out as described previously .
We previously reported that when primary CEM cultures were treated with 2% DMSO, the expression of muscle-specific proteins and nuclear lamin A was reversibly inhibited (Fig. 1, this paper; also, see ). Relative to control cultures of the same age, DMSO inhibited the incorporation of [35S]methionine into TCA precipitable proteins by 25 to 85%, the greater inhibition observed with longer exposure to DMSO. Following exposure to 2% DMSO for 5 days, the synthesis of muscle-specific proteins, such as desmin, skeletal muscle tropomyosins, and muscle-specific isoforms of the myosin light chains were greatly inhibited (compare control CEM in Fig. 1A to DMSO-treated CEM in Fig. 1B). The synthesis of a 26-kDa protein (~26) was, however, either induced by DMSO or, alternatively, enriched in DMSO-treated CEM cell homogenates (Fig. 1B) relative to control CEM (Fig. 1A) when [35S]methionine counts from longterm labelings were equilibrated. Despite the observation that DMSO treatment did not prevent the formation of myotubes, the 2D gel protein profile of the Day 5 DMSO-treated CEM appeared to resemble 24-h myoblasts (see ). Inclusion of cytosine fi-D-arabinofuranoside (ara-C) for 72 h in the DMSO treatment culture media (to eradicate any replicative cells) did not alter the protein labeling pattern (data not shown), indicating the DMSO-treated myotubes maintain the capacity for protein synthesis and that the resulting protein labeling patterns were not due to the preferential labeling of fibroblast and myoblast cells. DMSO Altered CEM Morphology DMSO treatment resulted in changes in the morphology of CEM cells as demonstrated in the scanning electron micrographs of Day 5 control and DMSO-treated CEM cells (Fig. 2). The majority of Day 5 control CEM (Fig. 2A) cells were syncytial, elongated, and well spread on the substratum. However, the DMSO-treated CEM cells exhibited myotubes with atrophied morphology (Fig. 2B). Immunofluorescence analysis for nuclear lamin A staining showed clusters of nuclei in the DMSOtreated myotubes (data not shown), indicating that DMSO treatment did not inhibit fusion of myoblasts. The observation of syncytial myotubes, which are refractory to the effects of ara-C, is consistent with the suggestion that DMSO-treated myotubes maintain the capacity for protein synthesis.
FIG. 1. Two dimensional gel analysis of control (A), and DMSO-treated (B) CEM cells. CEM cells were grown in culture for 5 days either in the absence (A) or in the presence of 2% DMSO (B), the last 12 h with 50 aCi/ml of [“S]methionine. The relative incorporation of [?S]methionine was determined by TCA precipitation and scintillation counting. Equal numbers of counts of each sample were subjected to 2D gel electrophoresis with pH 4-6 ampholines in the first dimension. Fluorographs of the gels are shown. The migration positions of various proteins are labeled in A. Abbreviations used: TN-C, troponin C; lc-1, lc-2, myosin light chains-l and -2, respectively; TM, tropomyosin, skeletal muscle (Y and 0, their phospborylated isovariants p-oc and p-8, and the nonmuscle isoform TM-l, which corn&rates with the skeletal muscle isoform cr-TM. p26 indicates the position of the 26-kDa protein described in the text.
p26 Is Identical
We tried various labeling schemes to determine the optimal p26 labeling conditions for further characterization and protein purification. Brief pulses of Day 7 CEM with [35S]methionine showed that relative to muscle-specific proteins p26 labeled to very high levels under both control and DMSO treatment conditions. Long-term labeling and Western blotting experiments, however, demonstrated that p26 did not accumulate to correspondingly high levels (for example, see Fig. 1A).
These results indicate that the high level labeling of stable sarcomeric structural proteins dilute out proteins which do not accumulate, such as ~26, and therefore do not appear in the homogenates of long-term-labeled control cell homogenates (for example, as in Fig. 1A). To determine if the lack of p26 accumulation was due to protein degradation or secretion, Day 7 CEM cell homogenate and culture medium were analyzed for the presence of p26 by pulse-chase experiments (Fig. 3). p26 was labeled to high levels by the 30-min pulse-label (Fig. 3, lane 1) and was immunoprecipitated by the anti-
FIG. 2. DMSO altered the morphology of cultured CEM cells. Scanning electron microscope (SEM) micrographs of control (A) and DMSO-treated (B) Day 5 CEM cells. The cultured cells were processed for SEM as described under Materials and Methods. The bar in B equals 0.76 mm.
munoprecipitate independently (data not shown). Based on the cellular, biochemical, and immunological characteristics of ~26, we conclude that the 26-kDa protein is identical to chicken apolipoprotein A-l and will henceforth refer to p26 as Apo A-l. in CEM Cells of 23 Min
Apo A-l Has a Half-Life
FIG. 3. p26 was labeled to high levels in [?S]methionine-pulsed CEM cells and secreted within a 3-h chase. Parallel cultures of Day 7 CEM cells were pulse-labeled for 30 min with 50 &i/ml of [?S]methionine, harvested immediately following the pulse (lanes 1 and 2), or chased with unlabeled methionine (5 mM) for 3 h before harvesting the cells (lanes 3 and 4) and culture medium (lanes 5 and 6). An equal percentage of the total homogenates and dialyzed medium (lanes 1,3, and 5) or the anti-p26 immunoprecipitates (lanes 2, 4, and 6) were analyzed by SDS-PAGE and fluorography. p26 indicates the position of the 26-kDa protein indicated in Fig. 1B. The migration positions of molecular weight markers are indicated on the left.
To determine the rate of Apo A-l turnover in CEM cells, a detailed pulse-chase analysis was carried out. As can be seen in Fig. 5A, pulse-labeled Apo A-l in cell homogenates decreased as the chase time progressed (lanes l-6). To quantify the amount of labeled Apo A-l in cell homogenates at each chase time point, we immunoprecipitated Apo A-l with rabbit anti-p26 serum (lanes 7-12). Again, the labeled Apo A-l decreased in the cell homogenate with the increase in chase time, such that only trace amounts were observed under the 240-min chase condition (lanes 6 and 12). By scintillation counting of the immunoprecipitates from each chase time point, we quantitated the rate of Apo A-l loss from the CEM cell homogenates (Fig. 5B). It appeared that Apo A-l has a t,,, in cell homogenates of 23 min. A slightly longer & (32 min) was obtained when
116.5 kD *
p26 antiserum (rabbit 7; lane 2). However, following the 3-h chase, the pulse-labeled p26 had decreased in the CEM cell homogenate (lane 3) and was nearly quantitatively observed in the chase culture medium (lane 5). Rabbit 7 antiserum immunoprecipitated only minor quantities of p26 from the 3-h chase CEM cell homogenate (lane 4) and large quantities from the chase culture medium (lane 6). From these results we concluded that p26 is a secreted protein. The cellular and biochemical properties of p26 suggested that p26 might be identical to chicken apolipoprotein A-l . To determine the identity of ~26, we labeled Day 7 CEM with [35S]methionine and immunoprecipitated the total cell homogenate with either antiserum against p26 (Fig. 4, lane 1) or a rabbit antiserum generated against purified chicken Apo A-l (Fig. 4, lane 2; generously provided by H. Lebherz, San Diego State University, San Diego, CA). Proteins of identical mobility were immunoprecipitated by the two antisera. The anti-p26 and anti-APO A-l immunoprecipitates were further characterized by 2D gel analysis. Mixing of equal counts from the immunoprecipitates of the two antisera generated a 2D pattern of proteins with isoelectric variants identical to the pattern generated by either im-
94 kD * 66 kD *
46 kD )
30 kD* ~26
21I kD *
FIG. 4. Identification of p26 as apolipoprotein A-l. Day 7 CEM cells were labeled for 5 h with 50 &i/ml of [%]methionine. The anti-p26 immunoprecipitate (lane 1) and anti-apolipoprotein A-l immunoprecipitate (lane 2) were analyzed by SDS-PAGE. The migration positions of molecular weight markers are indicated on the left. p26 indicates the position of the 26kDa protein indicated in Fig. 1B.
94 kD’ 68 kDr
30 kD* Ape A-l 21 kD’
Chase Time (min.) FIG. 5. Apolipoprotein A-l had a tin of 23 min in CEM cell homogenates. (A) Day 7 CEM cells were pulse-labeled for 30 min with 100 &i of [35S]methionine then chased with unlabeled methionine (final concentration 5 mA4) for 0 (lanes 1 and 7), 15 (lanes 2 and S), 30 (lanes 3 and 9), 60 (lanes 4 and lo), 120 (lanes 5 and ll), or 240 (lanes 6 and 12) min. Total cell homogenates (lanes l-6) or anti-p26 immunoprecipitates (lanes 7-12) were then analyzed by SDS-PAGE. The migration positions of molecular weight markers are indicated on the left. The spot labeled Apo A-l identifies the migration position of apolipoprotein A-l. (B) Quantitation of the anti-p26 immunoprecipitates shown in Fig. 5A 23 and plotting the cpm’s versus chase time. It was determined that apolipoprotein A-l has a tin in CEM cell homogenates of approximately min.
the half maximal rate of secretion for Apo A-l was determined from the immunoprecipitated cell media. Pulse times as short as 4 min gave essentially identical results (data not shown). Expression of Apo A-l in Differentiating
To characterize temporally the differential expression of Apo A-l and muscle-specific proteins in our culture system, cultures of CEM cells at various stages of differentiation were pulse-labeled for 30 min with [35S]methionine, and the total homogenates were analyzed by 2D gel electrophoresis (Fig. 6). The level of [35S]methionine incorporation into Apo A-l increased with time in culture, from a barely detectable amount in the 24-h culture homogenate (Fig. 6A) to a major protein seen in the 96-h (Fig. 6B) and 168-h (Fig. 6C) culture homogenates. The Apo A-l immunoprecipitated from CEM cells indicated that Apo A-l had quantitatively minor acidic isoelectric variants (see Fig. 6C). Efforts to identify the nature of the isoelectric shift by labeling Day 7 CEM with [32P]phosphate failed to label any of the Apo A-l isoelectric variants, suggesting that the isovariant was not modified by a phosphate group (data not shown; see ). This increase in the labeling of Apo A-l during in vitro muscle differentiation ap-
peared to follow the same temporal pattern as for the muscle-specific proteins (Y-and /I-tropomyosins, the intermediate filament protein desmin, and myosin light chains-l and -2 (see Figs. 1A and 6C for labels). Therefore, these results are consistent with the suggestion by Ferrari et al.  that Apo A-l expression may be differentially and coordinately regulated with muscle-specific proteins. We have, however, in our previous study , and in this study as described below, demonstrated that Apo A-l (~26) expression does not strictly follow muscle-specific protein expression. We show that Apo A-l expression was resistant to DMSO treatment, whereas under the same conditions the synthesis of muscle-specific proteins was greatly inhibited. DMSO Inhibits Muscle-Specific but Not Apo A-l Expression To further characterize the relationship between Apo A-l and muscle-specific protein expression, we examined the expression of Apo A-l in control, DMSOtreated, and DMSO-recovered CEM cells, and quantified Apo A-l abundance by immunoprecipitation (Fig. 7). In contrast to the DMSO inhibition of muscle-specific protein expression (see Fig. l), the synthesis of Apo A-l remained at approximately the same level whether
returned to high levels (lane 4), although somewhat lower than levels observed in control cells (lane 3), whereas cells maintained in DMSO synthesized little or no skeletal muscle tropomyosins (lane 5). However, the synthesis of Apo A-l was approximately equal in control, DMSO-recovered, and DMSO-treated CEM cultures (lanes 8-10, respectively).
DMSO Did Not Alter the Rates of Apo A-l Secretion Pulse-chase experiments were performed to determine if the rates of Apo A-l secretion differed in control and DMSO-treated CEM cells. Figure 8 shows that the decrease in the abundance of Apo A-l in the cell homogenates is inversely related to the amount of Apo A-l in the culture medium. Total [36S]methionine incorporation (TCA precipitable counts) into Day 7 control CEM cells (Fig. 8, lanes l-3) was approximately eightfold higher than the incorporation into DMSO-treated CEM cells (lanes 7-9). However, the incorporation of [35S]methionine into Apo A-l, as detected by immunoprecipitation of both the cell homogenate and the corresponding culture medium, was approximately equal in
200 I k0 116.5
94 k0 68
30 k0 APO / A.
FIG. 6. Apolipoprotein A-l was differentially expressed in cultured CEM. Cells were pulse-labeled for 30 min with 50 &i/ml of [?‘S]methionine at 24 h (A), 96 h (B), or 168 h (C) postplating. For 2D gel analysis, equal counts of each sample were loaded onto the first dimensional IEF gels (ampholines, pH 5-7). Note the appearance of the spot to the acidic side of Apo A-l in C. The position of specific proteins corresponding to those labeled in Fig. 1 are noted by arrowheads. The spot labeled Apo A-l identifies the migration position of apolipoprotein A-l.
the Day 5 CEM cells were cultured under control (Fig. 7, lanes 1 and 6) or DMSO treatment conditions (lanes 2 and 7). After removal of DMSO for 72 h, the synthesis of the muscle-specific proteins (e.g., muscle tropomyosin)
FIG. 7. DMSO treatment inhibited muscle-specific, but not Apo A-l, protein synthesis. Day 5 control (lanes 1 and 6) and 5&y DMSO-treated (lanes 2 and 7). or Day 8 control (lanes 3 and 8), 3-day recovered 5-day DMSO-treated (lanes 4 and S), and 8-day DMSOtreated (lanes 5 and 10) CEM cultures were pulsed-labeled for 30 min with 50 rCi/ml of [%]methionine. The total homogenates (lanes l-5) and the anti-p26 immunoprecipitates (lanes 6-10) were analyzed by SDS-PAGE. The sample volumes loaded onto SDS-PAGE gels represented an equivalent number of cells. Double arrowheads mark the migration positions of the skeletal muscle isoforms of a- and fl-tropomyosin (TM’s). The migration positions of molecular weight markers are indicated on the left.
control and DMSO-treated CEM at every label point. This indicated that the rate of secretion and, again, the rate of synthesis were equivalent for control and DMSO-treated CEM cells. In Vitro Translation To determine if the differential expression of Apo A-l observed during skeletal muscle development reflected changes in Apo A-l mRNA levels, total RNAs or poly(A)+ mRNAs were isolated from chick embryo fibroblasts, myoblasts, myotubes, and DMSO-treated myotubes. The RNA driven cell-free translation products (Fig. 9, lanes 1-5) and their anti-APO A-l immunoprecipitates (lanes 6-10) were examined by SDS-PAGE. As can be seen in Fig. 9, very low quantities of Apo A-l were detected in the translation products directed by both CEF (lanes 1 and 6) and myoblast (lanes 2 and 7) RNAs, whereas abundant amounts of Apo A-l were detected in the translation products directed by myotube (lanes 3 and 8) and DMSO-treated myotube (lanes 4 and 9) RNAs. The abundance of in vitro-translated Apo A-l largely reproduced the in viuo expression pattern of Apo A-l synthesis seen in differentiating CEM and DMSOtreated CEM cells. Poly(A)+ mRNAs also directed high levels of Apo A-l synthesis (lanes 5 and 10). Furthermore, the approximately equal amounts of Apo A-l that were immunoprecipitated from the translation products directed by myotube or DMSO-treated myotube RNAs suggest that RNA levels for Apo A-l were in equal abundance under both conditions.
FIG. 8. The rate of apolipoprotein A-l secretion was unaffected by the presence of DMSO. Control (lanes l-6) and DMSO-treated (lanes 7-12) Day 7 CEM cells were pulsed with 20 pCi of [“Slmethionine then chased for 0 min (lanes 1,4, 7, and lo), 30 min (lanes 2,5,8, and ll), or 60 min (lanes 3,6,9, and 12) with unlabeled methionine (final concentration 5 mM). Total cell homogenates (lanes l-3 and 7-9) and culture media (lanes 4-6 and 10-12) were collected and analyzed by scintillation counting and SDS-PAGE. The gel was loaded with 1% of the starting material of each sample. The migration positions of molecular weight markers are indicated on the left. The migration position of apolipoprotein A-l is identified.
FIG. 9. In vitro translation indicated that the abundance of Apo A-l mRNA was unaffected by DMSO. In vitro translation reactions of rabbit reticulocyte lysates in the presence of 10 pCi of [35S]methionine were carried out using 10 pg of total RNA isolated from chick embryo fibroblasts (CEF, lanes 1 and 6), 24-h myoblasts (lanes 2 and 7), Day 7 control CEM (lanes 3 and 8), Day 7 DMSOtreated CEM (lanes 4 and 9), or 1.0 fig of poly(A) mRNA isolated from control Day 7 CEM cells (lanes 5 and 10). Total homogenates (lanes 1-5) and anti-p26 immunoprecipitates (lanes 6-10) were analyzed by SDS-PAGE. The migration positions of molecular weight markers are indicated on the left.
DISCUSSION We previously reported the persistent synthesis of a 26-kDa protein during the DMSO treatment of CEM cells . We have now identified p26 as apolipoprotein A-l by the following criteria: immunological crossreactivity of p26 with anti-chicken Apo A-l antisera; identification of p26 as a secreted protein; and the identical molecular weights and comigration of p26 with Apo A-l in 1D and 2D gel analysis. In this study, we have further temporally characterized the pattern of Apo A-l expression during myogenesis. Apo A-l was synthesized at low levels in myoblasts and at high levels in CEM myotube cultures. Apo A-l, however, did not attain high levels of synthesis until after that of muscle-specific proteins. Thus, Apo A-l appeared to be differentially expressed in CEM cells, in a manner similar to the expression of muscle-specific proteins. We have, however, by DMSO treatment of CEM cells demonstrated that expression of Apo A-l can be uncoupled from the differential synthesis of muscle-specific proteins. The inhibition of cell fusion and transcription of both muscle-specific and Apo A-l genes by treatment of myoblasts with phorbol esters, or by Rous sarcoma virus infection, had been observed by Ferrari et al. . They thus suggested that expression of the Apo A-l gene was strictly linked and coordinately regulated with muscle-
specific gene expression . In this report, we have clearly demonstrated that DMSO inhibited muscle-specific but not Apo A-l gene expression. These results contradict the suggestion by Ferrari et al.  and apparently indicate that Apo A-l gene expression is not solely regulated by the pathways or mechanisms required for the expression of sarcomeric protein genes. We have not, however, determined the precise mechanism by which DMSO exerts its inhibitory effects on the expression of muscle-specific genes. There is accumulating evidence that during myogenesis the differential expressions of muscle-specific genes are dependent on the activity of skeletal muscle-specific tram-acting factors, the MyoD family (for review, see ). The MyoD family proteins have been shown to bind conserved sites present in the promoters and enhancers of muscle-specific genes, such as the MCAT, E-box, or CA& box motifs (for a description, see ). Sequence analysis of the 5’ flanking region of the chicken Apo A-l gene (351 failed to reveal the presence of conserved muscle gene regulatory elements. This is consistent with our suggestion that during myogenesis Apo A-l expression is regulated by mechanisms independent of those which regulate the expression of muscle-specific genes. Furthermore, it has been recently reported that in murine muscle cell lines, DMSO inhibits the insulin-like growth factor-l-dependent elevation of myogenin mRNA , a member of the MyoD family of skeletal muscle-specific DNA binding factors. The demonstration that DMSO inhibits the synthesis of the MyoD family of skeletal muscle factors in CEM cells would substantially strengthen our suggestion of independent and separate regulatory mechanisms. Additional support for separate regulatory mechanisms would be derived by the demonstration that other myogenie inhibitory agents do not interfere with the expression of Apo A-l, in particular, myogenic inhibitory agents which do not interfere with membrane fusion/ remodeling events. The observations that Apo A-l was transiently expressed in a variety of differentiating embryonic and adult chicken cell types [16-19,37,38] and that musclespecific genes continue to be expressed at high levels following the transient period of Apo A-l expression in the skeletal muscle of peripheral tissues [18, 191 also appear incompatible with the suggestion of Apo A-l expression being tightly linked to muscle-specific gene expression. The high rates of synthesis of Apo A-l observed in various tissues in ouo had suggested that up-regulation of Apo A-l expression may be due to the appearance of extracellular factors or lipids, as from yolk uptake which precedes hatch [17,19,21,37-411. However, if in ouo expression of Apo A-l were dependent solely on the occurrence of yolk lipid uptake, we would not anticipate that Apo A-l would be differentially expressed in the
defined and relatively constant extracellular environment maintained during in vitro CEM differentiation. The reported time course of the formation of the sarcoplasmic reticulum and T-tubule membrane systems in developing myotubes in ouo and in vitro appears to temporally parallel the pattern of Apo A-l expression [42-441, suggesting that Apo A-l expression may be associated with extensive membrane synthesis and/or remodeling events, such as formation of the T-tubule and sarcoplasmic reticulum membrane systems in skeletal muscle [42,43], and myelination of neurons [17,38]. In dystrophic chicken skeletal muscle, it is interesting to note that the failure to fully down regulate Apo A-l correlates well with excessive proliferation of the sarcolemma, sarcoplasmic reticulum, and T-tubule membrane systems, and their elevated cholesterol content [43, 45, 461. Whether a cause and effect relationship exists for membrane-mediated events with the transient pattern of Apo A-l expression in peripheral tissues is currently unknown. The expression of Apo A-l in chicken  and in mammals  appears to be regulated by multiple mechanisms which include gene induction and/or derepression. Recently, both positive- and negative-acting factors which modulate Apo A-l gene expression have been identified in human liver cells [35, 481. The negativeacting factor appears to be a steroid receptor , indicating a role for steroid-like molecules. In addition, different c&acting 5’ sequence elements control expression of the mammalian Apo A-l gene in liver and intestinal tissue cell types [49, 501, supporting the suggestion that multiple regulatory pathways, acting independently or in combination, direct the complex pattern of Apo A-l gene expression. The demonstration that Apo A-l is differentially expressed in a CEM culture system suggests that the CEM culture model is a useful system for examining Apo A-l function, as well as mechanisms of regulation of expression. Components of the culture medium, extracellular matrix, cell density, and type may be assayed for their effects on Apo A-l expression and peptide maturation, secretion, and HDL particle formation. Of particular interest would be the effects produced by specific lipids and glucocorticoids on Apo A-l expression or agents which specifically disrupt lipid metabolism and/or membrane synthesis/remodeling events during myogenesis. In addition, a more extensive description of the chicken Apo A-l gene regulatory elements, especially those which confer the apparently developmentally regulated expression during myogenesis, as well as characterization of the factors which bind such elements, will be of particular interest. We are indebted to J. L.-C. Lin and L.-P. Jin for technical assistance, to Dr. H. Lebherz (San Diego State University, San Diego, CA) for helpful discussions and generously supplying the rabbit antichicken Apo A-l serum, and to Dr. E. Spaziani, Kevin Cook, and
Lynn Leverenz for critical readings of the manuscript. This work is supported in part by grants HD18577 and GM40508 from the National Institutes of Health and by grants from the Muscular Dystrophy Association and the Pew Memorial Trust. Dr. J. J.-C. Lin is a recipient of a Pew Scholarship in Biomedical Sciences from the Pew Memorial Trust.
REFERENCES 1. Cossu, G., Ranaldi, G., Senni, M., Molinaro, (1988) Development 102,65-69. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13.
15. 16. 17. 18. 19. 20. 21. 22. 23.
M., and Vivarelli,
Endo, T., and Nadal-Ginard, B. (1987) Cell 49,515-526. Friend, C., Scher, W., Holland, J., and Sato, T. (1971) hoc. Natl. Acad. Sci. USA 68,378-382. Paulin-Lavasseur, M., Giese, G., Scherbarth, A., and Traub, P. (1989) Eur. J. Cell Biol. 50, 453-461. Van Roosendaal, K., Darling, D., and Farzaneh, F. (1990) Exp. Cell Res. 190, 137-140. Kimhi, Y., Palfrey, C., Spector, I., Barak, Y., and Littauer, U. (1976) Proc. Natl. Acad. Sci. USA 73,462-466.
Lin, J. J.-C., Matsumura, F., and Yamshiro-Matsumura, (1984) J. Cell Biol. 98,116-127.
Lin, J. J.-C., Lin, J. L.-C., Davis-Nanthakumar, E., and Lourim, D. (1988) Hybridoma 7,273-288. Laemmli, U. (1970) Nature 227,680-685. O’Farrel, P. Z. (1975) J. Biol. Chem. 250,4007-4021. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76,4350-4354. Bonner, W., and Laskey, R. (1974) Eur. J. Biochem. 46,83-88.
26. 27. 28. 29. 30.
Maniatis, T., Fritsch, E., and Sambrook, J. (1982) Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Lourim, D., and Lin, J. J.-C. (1989) J. Cell Bi01. 109,495-504.
Zannis, V., Karathanasis, S., Forbes, G., and Breslow, J. (1986) in Methods in Enzymology (Segrest, J. P., and Albers, J. J., Eds.), Vol. 128, pp. 690-712, Academic Press, Orlando.
Weintraub, H., Davis, R., Tapscott, S., Thayer, M., Krause, M., Benezra, R., Blackwell, T., Turner, D., Rupp, R., Hollenberg, S., Zhuang, Y., and Lassar, A. (1991) Science 251,761-766.
Miranda, A., Nette, E., Khan, S., Brockbank, K., and Schonberg, M. (1978) Proc. Natl. Acad. Sci. USA 76, 3826-3830. Blau, H., and Epstein, C. (1979) Cell 17,95-108. Lourim, D., and Lin, J. J.-C. (1990) in Biochemical and Structural Dynamics of the Cell Nucleus (Wang, E., Wang, J. L., Chien, S., Cheung, W.-Y., and Wu, C.-W., Eds.), pp. 27-42. Academic Press, San Diego. Manduca, P., Castagnola, P., and Cancedda, R. (1988) Dev. Biol.
Santoro, I., Yi, T.-M., 1944-1953.
Widom, R., Ladias, J., Kouidou, S., and Karathanasis, S. (1991) Mol. Cell. Biol. 11. 677-687. Florini, J., Ewton, D., and Roof, S. (1991) Mol. Endocrinol. 6, 718-724. Shackleford, J., and Lebherz, H. (1983) J. Biol. Chem. 268, 7175-7180. LeBlanc, A., Foldvari, M., Spencer, D., Breckenridge, W., Fenwick, R., Williams, D., and Mezi, C. (1989) J. Cell Biol. 109, 1245-1256.
Terada, M., Fried, J., Nudel, U., Rifkind, R., and Marks, P. (1977) Proc. Natl. Acad. Sci. USA 74,248-252. Chapman, M. (1980) J. Lipid Res. 21, 789-853. Scanu, A., Edelstein, C., and Shen, B. (1982) in Lipid Protein Interactions (Jost, P. C., and Griffith, 0. H., Eds.), pp. 259-316, Wiley, New York. Gotto, A., Pownall, H., and Havel, R. (1986) in Methods in Enzymology (Segrest, J. P., and Albers, J. J., Eds.), Vol. 128, pp. 3-41, Academic Press, Orlando. Soutar, A., Garner, C., Baker, H., Sparrow, J., Jackson, L., Go&to, A., and Smith, L. (1975) Biochemistry 14,3057-3064. Blue, M.-L., Ostapchuk, P., Gordon, J., and Williams, D. (1982) J. Biol. Chem. 267, 11,X1-11,159. Shackleford, J., and Lebherz, H. (1983) J. Biol. Chem. 258, 14,829-14,833. Rajavashisth, T., Dawson, P., Williams, D., Shackleford, J., Lebherz, H., and Lusis, A. (1987) J. Biol. Chem. 262,7058-7065. Ferrari, S., Tarugi, P., Drusiani, E., Calandra, S., and Fregni, M. (1987) Gene 60,39-46. Glomset, J. (1968) J. Lipid Res. 9, 155-167. Ferrari, S., Battini, R., and Cossu, G. (1990) Dev. Biol. 140, 430-436. Lin, J. J.-C., Chou, C.-S., and Lin, J. L.-C. (1985) Hybrio!oma 4, 223-242. Konigsberg, I. (1979) in Methods in Enzymology (Jakoby, W. B., and Pastan, I. H., Eds.), Vol. 58, pp. 511-527, Academic Press, New York.
Received May 21, 1991 Revised version received July 12, 1991
and Walsh, K. (1991) Mol. Cell. Biol.
Tarugi, P., Reggiani, D., Ottaviani, E., Ferrari, and Calandra, S. (1989) J. Lipid Res. 30, 9-22.
Lindsey, S., Benattar, J., Pronczuk, Exp. Biol. Med. 195,261-269.
Elshourbagy, N., Boguski, M., Liao, W., Jefferson, L., Gordon, J., and Taylor, J. (1985) Proc. Natl. Acad. Sci. USA 82, 82428246. Ezerman, E., and Ishikawa, H. (1967) J. Cell Biol. 35,405-420. Shackleford, J., and Lebherz, H. (1985) J. Biol. Chem. 260,288291.
42. 43. 44. 45.
Merlie, J., Buckingham, Dev. Biol. 11, 61-114. Sumnicht,
A., and Hayes, K. (1990) J.
M., and Whalen,
G., and Sabbadini,
R. (1977) Curr. Top.
R. (1982) Arch. Biochem. Biophys.
Bonilla, E., Samitt, C., Miranda, A., Hays, A., Salviati, G., DiMauro, S., Kunkel, L., Hoffman, E., and Rowland, L. (1988) Cell 54,447-452.
Papazafiri, P., Ogami, K., Ramji, D., Nicosia, A., Monaci, P., Cladaras, C., and Zannis, V. (1991) J. Biol. Chem. 266, 57905797. Ladias, J., and Karathanasis, S. (1991) Science 251, 561-565. Haddad, I., Ordovas, J., Fitzpatrick, T., and Karathanasis, S. (1986) J. Biol. Chem. 261, 13,26813,277.
48. 49. 50.
Sastry, K., Seedorf, Biol. 8, 605-614.
U., and Karathanasis,
S. (1988) CeU. Mol.