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1 57 2 58 3 59 4 Neuromuscular Disorders xx (2002) xxx–xxx 60 5 www.elsevier.com/locate/nmd 61 6 62 7 63 8 Glucocorticoid-mediated regulation of utrophin levels in 64 9 65 10 muscle fibers 66 11 67 12 Isabelle Courdier-Fruh, Lee Barman, Alexandre Briguet, Thomas Meier* 68 13 69 MyoContract Pharmaceutical Research Ltd., Klingelbergstrasse 70, CH-4056 Basel, Switzerland 14 70 15 71 16 72 17 Abstract 73 18 Previous studies on transgenic mice indicate that upregulation of utrophin may offer a potential treatment strategy for Duchenne 74 19 muscular dystrophy. We have analyzed the effect of the glucocorticoid 6a-methylprednisolone-21 sodium succinate on utrophin protein 75 20 levels using a cell-based assay with differentiated human myotubes derived from biopsies of healthy individuals or Duchenne muscular 76 21 dystrophy patients. We found that within 5–7 days 6a-methylprednisolone-21 sodium succinate increases utrophin protein up to ,40% in 77 22 both normal and -deficient myotubes compared to untreated control cultures. When analyzed in promoter–reporter assays 6a- 78 23 methylprednisolone-21 sodium succinate activated a utrophin promoter A-fragment but did not activate a utrophin promoter B-fragment. 79 Surprisingly, endogenous levels of utrophin mRNA in 6a-methylprednisolone-21 sodium succinate-treated muscle cells were unaltered 24 80 indicating that the utrophin-inducing effect of glucocorticoids may be a result of post-transcriptional mechanisms. We have also analyzed 66 25 81 glucocorticoids for their effect on utrophin protein levels and found that glucocorticoids in general are able to induce utrophin protein in 26 human myotubes. q 2002 Published by Elsevier Science B.V. 82 27 83 28 Keywords: Utrophin; Prednisolone; Glucocorticoid; Duchenne muscular dystrophy 84 29 85 30 86 31 1. Introduction complex and levels of serum creatine kinase were normal- 87 32 ized [6–10]. Moreover, high levels of utrophin expression in 88 33 A potential treatment strategy for Duchenne muscular non-muscle mouse tissue have no toxic effect [11]. Based on 89 34 dystrophy (DMD) is based on the hypothesis that utrophin, these animal experiments small-molecule induced upregu- 90 35 a dystrophin-related protein, might functionally replace the lation of utrophin appears to be a valid strategy for DMD 91 36 defective dystrophin. Accordingly, elevated levels of utro- therapy. 92 37 phin homogeneously expressed along the myofiber There are several ways how utrophin levels can be modi- 93 38 membrane would improve the stability and protect myofi- fied in muscle cells, all of which may influence the outcome 94 39 bers from mechanical damage during repeated muscle of approaches to pharmacologically regulate levels of utro- 95 40 contraction [1]. This hypothesis is supported by the struc- phin protein. For instance, transcription from two indepen- 96 41 tural similarity of dystrophin and utrophin [2] and the find- dently regulated promoters results in utrophin with 97 42 ing that in DMD muscle utrophin compensates to some different N-terminal sequences [12,13]. In addition, there is 98 43 extent for dystrophin deficiency [3]. Experimental evidence increasing evidence for post-transcriptional regulatory 99 44 that utrophin attenuates the effect of dystrophin deficiency mechanisms that contribute to the control of utrophin 100 45 stems from the analysis of utrophin-dystrophin deficient protein levels and distribution [14–16]. Finally, utrophin 101 46 mice (double mutants) which show a more severe muscle protein stability might be influenced by the activity of 102 47 dystrophy phenotype than the dystrophin-deficient mdx calcium activated cysteine proteases (calpains) [17]. 103 48 mouse [4,5]. In addition, by expressing utrophin Pharmacological treatment strategies for DMD are 104 49 constructs in dystrophic mdx-mice, it was possible to restore currently confined to the use of certain steroid hormones. 105 50 the mechanical stability of muscle cells. In these transgenic In particular, the beneficial effect of 6a-methylpredniso- 106 51 animals, a number of disease-relevant parameters such as lone-21 sodium succinate (PDN) in young DMD patients 107 52 muscle force and morphology, integrity, is widely documented. Long-term studies in randomized, 108 53 expression ofUNCORRECTED components of the dystrophin glycoprotein double-blind, controlledPROOF clinical trials revealed increased 109 54 muscle strength following treatment with glucocorticoids, 110 55 such as PDN or deflazacort [18–20]. Clearly, one mechan- 111 * Corresponding author. Tel./fax: 141-61-267-2108. 56 E-mail address: [email protected] (T. Meier). ism of action in DMD is the anti-inflammatory and immu- 112

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113 nosuppressive effect of PDN [21]. However, cell culture RU38486 (mifepristone, RU486; Sigma Chemicals 169 114 experiments revealed additional properties of PDN that M8046; [27]) referred to as RU486 hereafter, were prepared 170 115 might contribute to the beneficial outcome when applied in DMSO at concentrations of 10 mM. 171 116 in dystrophic muscle. For example, PDN attenuates cellular Utrophin protein determination was carried out routinely 172 117 calcium uptake and therefore normalizes calcium handling with a mouse monoclonal antibody to the aminoterminal 173 118 in mechanically stressed muscle cells from mdx mice [22]. portion of utrophin (NCL-DRP2, Novocastra Labora- 174 119 PDN may also directly influence calcium-dependent tories). For control purposes a polyclonal rabbit anti-utro- 175 120 protease activity [23] which appears to be elevated in phin antibody to the rod-domain of utrophin (anti-UT31; 176 121 dystrophic muscle due to the impaired membrane integrity [28]) was also used. In addition anti- antibody 177 122 [24]. (D8281; Sigma) and anti- antibody specific for 178 123 The best-characterized cellular response of glucocorti- slow and fast myosin isoforms (M7523; Sigma) were 179 124 coids is the regulation of gene transcription. Distinct protein also used for protein quantification. Secondary antibodies 180 125 domains characterize nuclear receptors including glucocor- were purchased from Jackson ImmunoResearch Labora- 181 126 ticoid receptors (GRs). Interaction of the glucocorticoid tories. 182 127 with the ligand binding domain (LBD) liberates the gluco- 183 128 corticoid receptor from interacting chaperones and ensures 184 129 selectivity of the physiological response. Upon activation 2.2. Cell culture 185 130 and translocation to the nucleus, glucocorticoid receptor 186 131 homodimers are recruited via their DNA-binding domains Primary human cultures were prepared from 187 132 to specific DNA sequences, the so-called glucocorticoid muscle biopsies taken during orthopedic surgery from 188 133 response elements (GREs), which typically are inverted Duchenne patients and donors with normal muscle physiol- 189 134 hexanucleotide repeat sequences separated by one to several ogy (provided by the Association Franc¸aise contre les 190 135 base pairs [25]. Upon DNA binding the glucocorticoid Myopathies and the Muskelbank Mu¨nchen). Cultures were 191 136 receptor interacts with the transcription apparatus and in prepared and maintained according to standard protocols 192 137 conjunction with specific transcription factors and co-acti- [29]. 193 138 vators regulates [26]. 194 139 In this study we have analyzed the effect of PDN on 195 140 utrophin protein content and transcriptional regulation in 2.3. Utrophin protein quantification 196 141 human muscle cells. During the course of these experiments 197 142 we identified a number of steroids with clear glucocorticoid- Normalized concentrations of utrophin protein in human 198 143 like activity that induced utrophin protein content in human muscle cells cultured in microtiter plates were determined 199 144 muscle cells to a similar extent as PDN. Results obtained by a cell-based enzyme-linked immunosorbent assay 200 145 with promoter–reporter activity assays indicated that PDN (ELISA) procedure that allows a successive readout for 201 146 can activate utrophin promoter A, although endogenous the cell density and utrophin protein level. For calibration, 202 147 levels of utrophin mRNA where not affected. Our experi- the cell density and differentiation was determined by absor- 203 148 ments suggest that glucocorticoid induced upregulation of bance measurements of the total dehydrogenase enzyme 204 149 utrophin protein may be an indirect event perhaps involving activity in each well using the colorimetric CellTiter 205 150 glucocorticoid-mediated inhibition of utrophin degradation. 96AQ One Solution Reagent Proliferation Assay (Promega) 206 151 according to the manufacturer’s recommendation. Subse- 207 152 quently, cells were fixed, washed, permeabilized with 208 153 2. Material and methods 0.5% (v/v) Triton X-100 and unspecific antibody binding 209 154 sites blocked by standard procedures. Utrophin protein 210 155 2.1. Reagents levels were determined immunologically with utrophin- 211 156 specific primary antibodies and with appropriate peroxi- 212 157 6a-Methylprednisolone-21 sodium succinate (referred to dase-coupled secondary antibodies using QuantaBlu 213 158 as PDN in this paper) was purchased from Pharmacia– Fluorogenic Peroxidase Substrate kit (Pierce) for detection. 214 159 Upjohn (Solu-Medrol) and 5 mg/ml stock solutions were Fluorescence measurements were carried out with a multi- 215 160 216 prepared in dimethyl sulfoxide (DMSO) as solvent. In label counter (Wallac) at lex ¼ 355 nm and at lem ¼ 460 161 total 66 glucocorticoids were obtained from MicroSource nm. The primary readout of this signal is presented in arbi- 217 162 Discovery Systems Inc. (Gaylordsville, CT, USA) and trary units. For calibration, the arbitrary units representing 218 163 prepared as 3 mg/ml stock solutions dissolved in DMSO. the relative utrophin protein content of each well was 219 164 In all experiments, effects measured in cultures treated with divided by the corresponding cell-titer absorbance value to 220 165 PDN or PDNUNCORRECTED analogs were compared to the effects correct for cell PROOF density. For comparison between experi- 221 166 measured in cultures treated with appropriate concentrations ments, the cell-titer corrected readout for utrophin protein 222 167 of DMSO alone (solvent controls). Stock solutions of the content in each well was expressed in per cent of solvent 223 168 progesterone and glucocorticoid receptor antagonist treated control cultures (set to 100%). 224 ARTICLE IN PRESS

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225 2.4. Constructs, transfections and real-time polymerase Overall levels of endogenous utrophin mRNA in human 281 226 chain reaction (PCR) muscle cells were determined with real-time PCR. For this, 282 227 oligonucleotide primers and probes were designed with the 283 228 The mouse UTR-promoter (A)–reporter construct (pGL2- Primer Express 1.0 software (ABI Prism; PE Biosystems). 284 229 mUPA) was generated by PCR using primers s71mUP 50- Utrophin amplicon: forward primer AGCTTTTGAGGCC- 285 230 GGTCAGCACCAACACTATTTG-30, as1157 mUP 50- GATTCA, reverse primer TTAACAAATGCAGAG- 286 231 GTGGAAAGCCCGACAAGATCC-30 and mouse genomic CACTGG and probe CCCAAGGAGACGACGCAGGT- 287 232 DNA as a template. The PCR product was inserted into CTACA; 18S rRNA amplicon: forward primer CCGGA- 288 233 pGL-2 basic (Promega) opened with NheI. The human GAGGGAGCCTGAG, reverse primer GGGTAAT- 289 234 UTR-promoter (A)–reporter construct (pGL3-hUPA) was TTGCGCGCCT and probe ACGGCTACCACATCCAAG- 290 235 generated by PCR using primers shUPA 50-AAGCTTG- GAAGGCA. The probes purchased from Eurogenetec S.A. 291 236 GAACAGGCTCTATAACAG-3 0, ashUPA 50-CTCTAG- (Belgium) consisted of an oligonucleotide labeled at the 50- 292 237 GAGTTTGACACGGAGTAAC-30 and human genomic end with the reporter dye FAM and at the 30-end with the 293 238 DNA as a template. The PCR product was subcloned into quencher TAMRA. RNA was prepared from cells using 294 239 pGEM-T easy vector (Promega). Subsequently, the RNeasy mini kit (Qiagen). Random hexamers primed 295 240 hUPA fragment was excised using HindIII and inserted reverse transcription (RT) was performed using Superscrip- 296 241 into pGL-3 basic opened with HindIII. The human UTR- tII (Gibco BRL) using 1 mg total RNA according to manu- 297 242 promoter (B)–reporter construct was generated by PCR facturer’s recommendations. Real-time PCR was performed 298 243 using primers shUPB 50-GATTGTGGTGATGGTTGTA- with the TaqMan PCR Core Reagent kit (PE Applied 299 244 GAA-30, ashUPB 50-GAGATGAGGAAAAAGATGTG- Biosystems). The PCR mixture contained 500 nM of 300 245 GAG-30 and human genomic DNA as a template. The gene-specific primers, 200 nM of probe, and 1/10 of the 301 246 PCR product was inserted into pGL3-basic opened with RT reaction for the utrophin PCR or 1/100 of the RT reac- 302 247 SmaI. tion for the 18S rRNA reaction. Triplicate assays were 303 248 To measure the impact of different substances on the performed with cDNA samples generated from three inde- 304 249 glucocorticoid receptor pathway, transfection experiments pendent cultures for each condition. Amplification and 305 250 were performed in human myoblasts seeded at a density of detection of specific products was performed with the ABI 306 251 75 000 cells/well in six-well tissue culture plates. Twenty- Prism 7700 sequence detection system (PE Applied Biosys- 307 252 four hours after seeding, cells were transfected using the tems) with the following cycle profile: one step at 50 8C for 308 253 FUGENE 6 transfection reagent (Roche Diagnostics 2 min, one step at 95 8C for 10 min, 40 cycles at 95 8C for 15 309 254 310 Corp.) according to the manufacturer’s recommendations. s, and 60 8C for 1 min. The threshold cycle (CT)isdefined as 255 For each well, 1 mgofpDTAT3-LUC [30] and 0.2 mgof the cycle at which the fluorescence becomes detectable 311 256 p6R-LacZ [31] were co-transfected. Twenty-four hours later above background and is inversely proportional to the loga- 312 257 the medium was changed to medium containing test rithm of the initial number of template molecules. Utrophin 313 258 314 substances and cells were incubated for another 18–24 h. gene expression was calculated with the comparative CT 259 Cells were then extracted with Reporter Lysis Buffer method (ABI Prism 7700 sequence detection system, user 315 260 (Promega) and luciferase activity was determined using bulletin #2) using the 18S rRNA amplicon as an endogenous 316 261 the Luciferase Assay Reagent (Promega). Luminescence control. 317 262 was measured for 10 s after injection of luciferase reagent 318 263 using a Microlite TLX1 luminometer (Dynatech Labora- 319 264 tories Inc.). Values were normalized to the b-galactosidase 3. Results 320 265 activity measured in the extract based on the hydrolysis of 321 266 the substrate chlorophenol red–b-d-galactopyranoside 3.1. PDN increases utrophin protein levels in human muscle 322 267 (Boehringer Mannheim) as described [32]. cells 323 268 To measure the activation of utrophin promoter A or B, 324 269 COS-7 cells were transfected using the procedure describe To monitor levels of utrophin protein in human muscle 325 270 above. COS-7 cells were seeded in 24-well plates (20 000 cells, cell cultures enriched in myogenic cells were obtained 326 271 cells/well). Twenty-four hours later, the medium was from muscle biopsies taken from DMD patients and indivi- 327 272 replaced with transfection medium containing 10% char- duals with no muscle pathology (referred to as normal 328 273 coal-stripped FCS and cells were transfected with either controls). Myotubes were cultured in microtiter plates and 329 274 200 ng/well of pGL2-mUPA or pGL3-hUPA or pGL3- treated with PDN or an appropriate solvent control at the 330 275 hUPB and 300 ng/well of p6R-GR [33] and 100 ng/well time low-serum medium was added to induce myotube 331 276 of p6R-LacZ. Twenty-four hours after transfection, the differentiation. Cultures were maintained without further 332 277 medium was replacedUNCORRECTED with transfection medium containing changes of the mediumPROOF until myotubes formed, typically 333 278 the test substances and cells were incubated for another 18– 5–7 days. At the end of the experiment, cell titers were 334 279 24 h. The b-galactosidase and luciferase activities were determined by measuring total cell dehydrogenase enzyme 335 280 measured as describe above. activity in a colorimetric assay while levels of utrophin 336 ARTICLE IN PRESS

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337 protein were determined in the same wells using a cell- 393 338 based ELISA with utrophin specific antibodies. When 394 339 compared to DMSO treated cultures we observed elevated 395 340 levels of utrophin protein in normal muscle cultures treated 396 341 with 500 nM PDN (Fig. 1A). Measurement of mitochondrial 397 342 enzyme activities in the same well indicated a 10–15% 398 343 reduction in the overall cell titer upon PDN treatment 399 344 (Fig. 1B). For comparison between experiments the readout 400 345 for utrophin protein was normalized with the cell titer values 401 346 taken from the same well (Fig. 1C) and expressed as percent 402 347 induction with culture wells exposed to DMSO control set to 403 348 100%. Following this protocol, we routinely observed a 30– 404 349 40% increase in normalized utrophin protein levels in 405 350 normal muscle cultures (Fig. 1D) and a similar increase in 406 351 DMD muscle cell cultures (Fig. 1E). The effect of PDN on 407 352 normalized utrophin levels was dose-dependent with a 408 353 maximal effect seen at concentrations as low as ,500– 409 354 800 nM (Fig. 1F). The Utrophin-inducing effect of PDN 410 355 could be determined with antibodies recognizing the amino- 411 356 terminal portion of utrophin (DRP2, this antibody was used 412 357 routinely) as well as with a polyclonal antibody raised 413 358 against the mid-rod domain of utrophin (anti-UT-31 [28]; 414 359 Fig. 2A). The effect of PDN on muscle cell proteins was not 415 360 confined to utrophin. Instead we found that incubation of 416 361 myotubes with 500 nM PDN increased the levels of desmin 417 362 and fast and slow isoforms of myosin as well (Fig. 2B). This 418 363 result is consistent with a differentiation inducing effect 419 364 caused by incubation with the glucocorticoid. However, 420 365 our experiments did not address the question whether 421 366 PDN accelerates the formation of a small number of excep- 422 367 tionally well differentiated myotubes or increases the over- 423 368 all fusion index. 424 369 425 370 3.2. PDN-mediated utrophin induction in muscle cells is 426 371 dependent on glucocorticoid receptor activation 427 372 428 373 To address the question whether PDN can activate gluco- 429 374 corticoid receptor pathways in cultured human muscle cells 430 Fig. 1. 6a-Methylprednisolone-21 sodium succinate (PDN) increases utro- 375 431 we have monitored glucocorticoid receptor activation using phin protein levels in human muscle cells cultured in microtiter plates. (A) 376 a promoter–reporter construct. For this, normal muscle cells Normal muscle cell cultures were incubated with 500 nM PDN for 5 days and 432 377 were transfected with a plasmid encoding for three repeats levels of utrophin protein determined with a cell-based ELISA method using 433 378 of the glucocorticoid response element of the tyrosine a monoclonal antibody specific for the aminoterminal portion of utrophin. 434 , 379 aminotransferase in front of a minimal promoter (alcohol PDN-treated cultures showed a 20% increase in the utrophin-specific 435 signal (given in arbitrary units) when compared to cultures that were exposed 380 436 dehydrogenase) and luciferase reporter gene (pDTAT3-Luc; to solvent (DMSO) controls. (B) Cell titer measurements (expressed as 381 [30,32] and cotransfected with a LacZ plasmid for normal- absorbance units) in the same wells indicated a drop in overall cell density 437 382 ization. Stimulation of transfected muscle cells with PDN and/or myotube differentiation upon PDN treatment. (C) Normalized values 438 383 for 18–24 h revealed an 8-fold increase in TAT3-luciferase of utrophin protein levels (expressed as arbitrary units/absorbance) derived 439 384 activity compared to transfected but unstimulated control from the data presented in (A) and (B). (D) Same data as in (C) presented in 440 percent with control set to 100%. Using this method a ,45% increase of 385 441 cultures (Fig. 3A). PDN-mediated activation of this normalized utrophin protein levels were detected in normal muscle cells 386 TAT3-Luc reporter construct is prevented by addition of upon PDN treatment. (E) As with normal muscle cells, PDN also induces 442 387 the glucocorticoid receptor antagonist RU486 while appli- a ,33% increase in normalized utrophin protein levels upon PDN treatment 443 388 cation of RU486 alone had no effect on glucocorticoid in DMD muscle cells. (F) Dose–response relation of PDN-induced utrophin 444 ^ 389 receptor promoterUNCORRECTED–reporter activity (Fig. 3A). This result protein levels. Data PROOF expressed as mean standard deviation; (A–E) data 445 taken from 18 wells (PDN) or nine wells (control) in one microtiter plate 390 446 indicates that PDN can activate a glucocorticoid response for each bar/data point shown. (A–E) Asterisk indicates significant differ- 391 element in human muscle cells and that a glucocorticoid ence between PDN-treated and control cultures as determined by unpaired t- 447 392 receptor antagonist can suppress this activation. In addition, test, P , 0:0001. F: n ¼ 9 for each data point. 448 ARTICLE IN PRESS

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449 505 450 506 451 507 452 508 453 509 454 510 455 511 456 512 457 513 458 514 459 515 460 516 461 517 462 518 463 519 464 520 465 521 466 522 467 523 468 524 469 525 470 526 471 527 472 528 473 529 474 530 475 531 476 532 477 Fig. 2. Effect of PDN on utrophin, desmin and myosin levels in human muscle cells. (A) PDN-mediated increase of utrophin protein determined with antibodies 533 478 to the aminoterminal portion (DRP2) and mid rod-domain (UT-31) of utrophin. Top: schematic diagram representing the binding region of DRP2 and UT-31 to 534 distinct regions of the utrophin protein. Bar histogram: experiment as in Fig. 1 reveals a comparable level of utrophin increase following 500 nM PDN 479 535 treatment of normal muscle cells. Data expressed as mean ^ standard deviation; n ¼ 5. Asterisk indicates significant difference between PDN-treated and 480 control cultures (DMSO-treated) as determined by unpaired t-test, P , 0:0002. (B) PDN treatment increases the levels of utrophin, desmin, and myosin protein 536 481 in normal muscle cells (experiment as in Fig. 1). Following treatment with 500 nM PDN, protein levels for utrophin (left), desmin (center) and myosin (right) 537 W 482 were determined using appropriate primary antibodies. Data expressed as mean ^ standard deviation; n ¼ 12 for each bar. *P , 0:0001 and P , 0:001 538 483 (unpaired t-test) for PDN-mediated protein induction compared to control. 539 484 540 485 application of RU486 was able to inhibit PDN-mediated this assay system we detected a moderate increase in 541 486 induction of normalized utrophin protein levels in normal normalized luciferase activity upon PDN treatment with 542 487 and DMD muscle cells in culture (Fig. 3B,C). utrophin promoters A but not with utrophin promoter B. 543 488 Specifically, PDN incubation increased the level of normal- 544 489 3.3. PDN can activate utrophin promoter A in human ized luciferase activity up to 2.5-fold in experiments where 545 490 muscle cells the mouse utrophin promoter A (mUPA) fragment was used, 546 491 and up to 1.5-fold induction in experiments where the 547 492 We next tested whether the PDN-mediated increase in human utrophin promoter A (hUPA) fragment was analyzed 548 493 utrophin protein content was a consequence of transcrip- (Fig. 4A,B). In contrast, we did not detect activation of the 549 494 tional control from one of the two known utrophin promo- human utrophin promoter B (hUPB) fragment by PDN (Fig. 550 495 ters. To address this question we analyzed fragments of the 4C). The result that PDN can activate utrophin promoter A 551 496 mouse and human versions of utrophin promoter A and was supported by the result obtained from a computational 552 497 human utrophin promoter B in promoter–reporter assays. analysis. Using the NUBIScan algorithm [34], a weighted 553 498 For this, COS cells were transfected with either a 1086 bp matrix-based in silico approach, we have analyzed the 1514 554 499 fragment of mouse utrophin promoter A, or a 1239 bp frag- bp fragment of the mouse utrophin promoter and first exon 555 500 ment of human utrophin promoter A, or a 1464 bp fragment (GenBank accession number: X95524.1) where we identi- 556 501 of the human utrophinUNCORRECTED promoter B each cloned in front of a fied a nuclear receptorPROOF binding site arranged as an inverted 557 502 luciferase reporter. To allow for normalization cells were hexanucleotide repeat separated by a single base (AGGA- 558 503 cotransfected with two additional plasmids encoding for CAcTGACCT) at position 601–615. 559 504 glucocorticoid receptor [33] and LacZ reporter gene. With We next asked whether endogenous levels of utrophin 560 ARTICLE IN PRESS

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561 mRNA in muscle cells are influenced by PDN treatment. 617 562 For this, real-time PCR quantification of utrophin mRNA 618 563 levels in human muscle cells was carried out following 12, 619 564 24, 48 or 96 h of treatment with 500 nM PDN. As shown in 620 565 Fig. 4D, we did not detect elevated overall levels of utrophin 621 566 mRNA in muscle cells treated with PDN at any of the time 622 567 points measured. This indicated that PDN was able to 623 568 induce isolated utrophin promoter A/reporter constructs 624 569 but did not influence the endogenous levels of utrophin 625 570 mRNA. 626 571 627 572 3.4. PDN analogs increase utrophin protein levels in human 628 573 muscle cells 629 574 630 575 In the course of these experiments we analyzed in total 66 631 576 glucocorticoids and structural analogs of PDN for their 632 577 effect on utrophin protein levels in human muscle cells. 633 578 The chemical modifications were at positions 21 and 20 634 579 (R1–R3), position 17a (R4), positions 16 and 17 (R5, R6, 635 580 R8), position 11b (R7) and positions 9a and 6a (X and Y) 636 581 (Fig. 5). The double bond between C(4) and C(5) in ring A 637 582 as well as the keto-group in 3-position were maintained, 638 583 both of which are hallmarks of glucocorticoids. All 639 584 compounds were analyzed at concentrations of 500 nM 640 585 each and compared to PDN for their utrophin protein indu- 641 586 cing effect. All tested compounds induced utrophin protein 642 587 levels between 85 and 125% of the levels induced by the 643 588 standard corticosteroid PDN. Among the analogs that 644 589 showed the highest efficacy (Fig. 6A) were Triamcinolone 645 590 acetonide (code: 2-B9), Halcinonide (code: 2-D10), 646 591 Dichlorisone (code: 5-A9), and Fluocinolone acetonide 647 592 (code: 2-A4). When tested for glucocorticoid-like activity 648 593 using the TAT3–luciferase assay, all four compounds did 649 594 activate the glucocorticoid response element confirming 650 595 their glucocorticoid hormone activity (Fig. 6B). These 651 Fig. 3. The glucocorticoid receptor antagonist RU486 prevents PDN- PDN analogs were at least as efficient as PDN in increasing 596 induced utrophin protein increase. (A) PDN can activate a prototype gluco- 652 597 corticoid response element in human muscle cells monitored with a promo- utrophin protein levels in normal human muscle cells (Fig. 653 598 ter–reporter construct. Luciferase activity was measured in transfected 6C) or DMD muscle cells (Fig. 6D). All four compounds are 654 human muscle cells carrying the TAT3-Luc construct and a LacZ plasmid 599 known as highly potent glucocorticoids [35–38] and there- 655 for normalization. After transfection of normal human muscle cells with the 600 fore the effect seen on utrophin protein levels is probably a 656 two plasmids for 24 h, 500 nM PDN was applied for 24 h in charcoal- consequence of their hormone-like activity. Taken together, 601 stripped medium. When compared to unstimulated controls, PDN induced 657 602 normalized luciferase activity ,8-fold while incubation of cultures with this experiment shows that PDN and PDN analogs can 658 603 500 nM RU486 had no effect. Simultaneous application of 500 nM PDN increase the level of utrophin protein in human myotube 659 together with an equal concentration of RU486 completely abolished the 604 cultures. 660 PDN-mediated activation of the luciferase-coupled glucocorticoid response 605 element. Data expressed as mean ^ standard deviation. n ¼ 6 for each bar. 661 606 *P , 0:0001 (unpaired t-test) for PDN-induction compared to control; 4. Discussion 662 W 607 P , 0:0001 (unpaired t-test) for RU486-dependent inhibition compared 663 608 to PDN. (B,C) RU486 prevents PDN-mediated increase in normalized In this study we have analyzed the effect of PDN and 664 utrophin protein levels in normal human muscle cells (B) and DMD muscle 609 665 cells (C) while having no effect when applied alone to cultured muscle other glucocorticoids on the amount of utrophin protein 610 cells. Experiment as in Fig. 1, where normalized utrophin protein levels levels in human muscle using a cell-based ELISA approach. 666 611 were determined in PDN treated cultures and in cultures treated with 500 Our experiments demonstrate that PDN causes a ,35% 667 612 nM RU486 alone or with 500 nM RU486 in addition to 500 nM PDN. For increase in the level of normalized utrophin protein within 668 613 both cell types, RU486 inhibited the PDN-mediated increase in utrophin 669 UNCORRECTED^ 5–7 days starting PROOF at the time myotube fusion is initiated. A protein levels. Data expressed as mean standard deviation. n ¼ 9 for 614 670 each bar. *P , 0:0001 (unpaired t-test) for PDN-mediated utrophin protein similar observation was made earlier in primary cultures 615 induction compared to control; WP , 0:0001 for RU486-mediated inhibi- derived from normal and mdx mouse muscle [39]. In addi- 671 616 tion of utrophin protein induction compared to PDN. tion, it was reported that application of another synthetic 672 ARTICLE IN PRESS

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673 729 674 730 675 731 676 732 677 733 678 734 679 735 680 736 681 737 682 738 683 739 684 740 685 741 686 742 687 743 688 744 689 745 690 746 691 747 692 748 Fig. 4. PDN can mediate transcriptional activation of utrophin promoter A fragments but has no influence on the overall cellular levels of utrophin mRNA. (A) 693 749 Utrophin promoter fragment–luciferase assay using a 1086 bp fragment of the mouse utrophin promoter A (mUPA) in COS cells cotransfected together with 694 plasmids encoding glucocorticoid receptor and LacZ. Following a 24-h stimulation with 500 nM PDN, cells were harvested and analyzed for luciferase activity. 750 695 Normalized luciferase activity of PDN-treated cells is expressed as fold induction over untreated but transfected controls. (B) Experiment as in (A) with a 1230 bp 751 696 fragment of the human utrophin promoter A (hUPA). (C) Experiment as in (A) with a 1464 bp fragment of the human utrophin promoter B (hUPB). While PDN 752 697 moderately induces transcriptional activity of the utrophin promoter A fragments, no such induction was seen with utrophin promoter B fragments. Data 753 expressed as mean ^ standard deviation of normalized luciferase activity; n ¼ 3 for each bar; *P , 0:0001, WP , 0:01 (unpaired t-test) for PND-treated 698 754 cells compared to untreated controls. (D) PDN-treatment of muscle cells does not increase the overall level of endogenous utrophin mRNA as determinedby 699 real-time (TacMan) PCR. DMD muscle cells were cultured in medium supplemented with 500 nM PDN and mRNA levels were determined 12, 24, 48 and 96 h 755 700 later. Data (mean ^ standard deviation; n ¼ 3 independent wells for each bar) expressed as fold induction compared to controls (DMSO-treated cells). 756 701 757 702 glucocorticoid, dexamethasone, to human muscle cells for 758 703 prolonged periods of time (15 days) also induces utrophin 759 704 protein, although only in DMD but not in normal myotubes 760 705 [40]. Such a disease-specific effect was not confirmed in this 761 706 study where PDN increased normalized utrophin protein 762 707 levels in both normal and DMD muscle cell cultures. 763 708 Our results show that upon PDN treatment not only utro- 764 709 phin protein but also other myogenic markers such as 765 710 myosin and desmin are elevated to a similar extent. One 766 711 obvious explanation would be that PDN increases the 767 712 number and size of myotubes and therefore the overall 768 713 amount of these proteins, as discussed earlier [41,42]. 769 714 However, in our experiments the cell titer levels actually 770 715 were reduced upon PDN-treatment indicating that there 771 716 were fewer cells surviving in each well. Nevertheless, a 772 717 contribution of the accelerated myotube differentiation by 773 718 PDN as found in earlier experiments [43] cannot be 774 719 excluded. 775 720 Alternatively, PDN may increase myotube proteins 776 721 including utrophin by stimulating de novo synthesis via 777 722 regulation at the gene transcription level. Indeed, our 778 723 study revealed that PDN activates transcription from utro- 779 724 phin promoter A when analyzed in promoter–reporter 780 725 assays. In contrast,UNCORRECTED no such activation was observed with Fig. 5. PDN analogs assayedPROOF for their influence on utrophin protein levels in 781 human muscle cells. In total 66 glucocorticoids and PDN analogs were 726 a functional utrophin promoter B fragment. However, our 782 studied with a combination of structural variations indicated as R1–R8, 727 experiments also demonstrated that in differentiated and X, and Y. All compounds were tested at concentrations of ,500 nM 783 728 PDN-treated myotubes the overall utrophin mRNA levels dissolved in DMSO and compared to the effect of 500 nM PDN. 784 ARTICLE IN PRESS

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785 841 786 842 787 843 788 844 789 845 790 846 791 847 792 848 793 849 794 850 795 851 796 852 797 853 798 854 799 855 800 856 801 857 802 858 803 859 804 860 805 861 806 862 807 863 808 864 809 865 810 866 811 867 812 868 813 869 814 870 815 871 Fig. 6. Activity of selected PDN analogs. (A) Chemical structure of the PDN analogs with highest efficacy in utrophin protein induction analyzed in human 816 muscle cells. For each compound, the code, chemical name, trade name and source is provided. (B) Activation of a prototype glucocorticoid response element 872 817 by PDN analogs. Luciferase activity was measured in transfected COS cells carrying the TAT3-Luc construct and plasmids encoding for glucocorticoid 873 818 receptor and LacZ. When compared to DMSO-treated control cells (Con.), the four PDN analogs 2-A4, 2-B9, 2-D10, 5-A9 induced luciferase activity to 874 ^ ¼ : K : W : 819 comparable levels as PDN. Data expressed as mean standard deviation. n 3 for each bar. *P , 0 0001, P , 0 0003, P , 0 02 (unpaired t-test) for 875 normalized luciferase activity compared to control. (C,D) Effect of PDN analogs 2-A4, 2-B9, 2-D10, 5-A9 on normalized utrophin protein levels measured in 820 876 normal human muscle cells (C) or DMD muscle cells (D). Experimental design as in Fig. 1. Data expressed as mean ^ standard deviation. n ¼ 11 for 821 Fluocinolone acetonide (2-A4), Triamcinolone acetonide (2-B9), Halcinonide (2-D10), Dichlorisone (5-A9), n ¼ 33 for PDN and control. *P , 0:0001 877 822 (unpaired t-test) for glucocorticoids compared to control. All four PDN analogs induce utrophin protein levels to a similar extent as PDN. Note: the differences 878 823 between absolute utrophin levels between normal and DMD muscle cells indicate experimental variation. In general, utrophin protein induction by PDN as well 879 824 as PDN analogs was not different in normal muscle cells compared to DMD muscle cells. 880 825 881 are unaltered, indicating that utrophin promoter A may not Cell culture experiments demonstrated that PDN treatment 826 882 be accessible to glucocorticoids in differentiated myotubes. for 3–5 days protects muscle cells from elevated levels of 827 883 Indeed, binding affinities of the glucocorticoid ligand free cytosolic calcium concentrations [22], an effect that can 828 884 complex to the DNA binding site is dependent on the chro- be inhibited by RU486 [46]. In conclusion, PDN might 829 885 matin structure and accessibility of target GRE sequences inhibit calpain activity and consequently calcium-dependent 830 886 [44]. Glucocorticoid-mediated increase in utrophin protein protein breakdown. Although both dystrophin and utrophin 831 887 levels might also be regulated directly on the protein level can be cleaved by calpains as indicated from biochemical 832 888 without involvement of utrophin gene transcription. Possi- experiments [17,47], it remains to be determined to what 833 889 ble mechanisms include protection from protein degrada- extent calpain-mediated protein degradation contributes 834 890 tion. For instance, it has been shown that the calcium- directly to the turnover of utrophin to account for the 835 891 dependent cysteine protease calpain is activated in observed effect. 836 892 dystrophic muscle fibers [45] and that PDN at high concen- Interpretation of these results is further complicated by 837 893 trations directlyUNCORRECTED inhibits calpain in vitro [23]. In addition, observations thatPROOF glucocorticoids also stimulate protein 838 894 PDN might influence protein breakdown indirectly by breakdown both in vivo and in cultured muscle cells. For 839 895 normalizing the calcium homeostasis in dystrophic muscle. instance, dexamethasone activates protein degradation in 840 896 ARTICLE IN PRESS

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897 cultured myotubes by inducing transcription of cathepsin [13] Burton EA, Tinsley JM, Holzfeind PJ, Rodrigues NR, Davies KE. A 953 898 and calpain proteases [48]. Interestingly, this glucocorti- second promoter provides an alternative target for therapeutic up- 954 regulation of utrophin in Duchenne muscular dystrophy. Proc Natl 899 coid-mediated induction of protease mRNA can be 955 Acad Sci USA 1999;96:14025–14030. 900 956 prevented by RU486. In summary, the role of glucocorti- [14] Vater R, Young C, Anderson LV, et al. Utrophin mRNA expression in 901 coids such as PDN and PDN analogs in the regulation of muscle is not restricted to the . Mol Cell 957 902 protein content and turnover appears to be complex. There- Neurosci 1998;10:229–242. 958 903 fore, further studies will be necessary to understand the [15] Gramolini AO, Karpati G, Jasmin BJ. Discordant expression of utro- 959 904 possible mechanisms relevant for the pharmacological regu- phin and its transcript in human and mouse skeletal muscles. J Neuro- 960 pathol Exp Neurol 1999;58:235–244. 905 961 lation of therapy-relevant components such as utrophin. [16] Gramolini AO, Belanger G, Jasmin BJ. Distinct regions in the 30 906 untranslated region are responsible for targeting and stabilizing utro- 962 907 phin transcripts in cells. J Cell Biol 2001;154:1173– 963 908 Acknowledgements 1183. 964 909 [17] Earnest JP, Santos GF, Zuerbig S, Fox JE. Dystrophin-related protein 965 in the platelet membrane skeleton. Integrin-induced change in deter- 910 We are grateful to J. Kalvoda for his help with the inter- 966 gent-insolubility and cleavage by calpain in aggregating platelets. J 911 pretation of the chemistry data. We also thank M. Podvinec 967 for computing, D. Knutti and A. 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