Ubiquitin gene expression is increased in human muscle undergoing neurogenic involvement

NEUROCHEMISTRY International Neurochemistry International 23 "0888# 026Ð039

Ubiquitin gene expression is increased in human muscle undergoing neurogenic involvement M[ Lloveraa\ C[ Garc(a!Mart(neza\ N[ Agellb\ F[J[ Lopez!Sorianoa\ F[J[ Authierc\ R[K[ Gherardic\ J[M[ Argilesa\ a

Departament de Bioqu(mica i Biolo`ia Molecular\ Facultat de Biolo`ia\ Universitat de Barcelona\ Barcelona\ Spain b Departament de Biolo`ia Cellular\ Facultat de Medicina\ Universitat de Barcelona\ Barcelona\ Spain c Departement de Patholo`ie\ Ho¼pital Henry Mondor\ Creteil\ France Received 8 April 0887^ received in revised form 2 September 0887^ accepted 05 November 0887

Abstract Histological features of neurogenic muscle involvement included type grouping\ muscle _ber atrophy\ and target _bers[ In muscles with myo_ber atrophy and target _bers\ we found an increased expression of the genes encoding for the ubiquitin!ATP! dependent proteolytic system[ Thus\ in patients with target _bers\ a 4[1! and a 2[8!fold increase were observed for the 1[3 and 0[1 kb transcripts\ respectively\ while in those with atrophic angulated hyperoxidative _bers\ a 2[8! and a 3[3!fold increase were observed for the 1[3 and 0[1 kb transcripts\ respectively[ It is suggested that the activation of this proteolytic system may be responsible for the skeletal muscle alterations that often accompany human muscle neurogenic involvement[ Þ 0888 Elsevier Science Ltd[ All rights reserved[ Keywords] Muscle denervation^ Hyperoxidative _bers^ Proteolysis^ Ubiquitin

0[ Introduction Di}erent experimental models have shown that increased muscle protein degradation seems to be the key factor involved in the loss of muscle weight in several patho! logical conditions including cancer cachexia\ infection\ acidosis\ and denervation atrophy "see Argiles and Lopez!Soriano "0885# for review#[ Recent progress\ con! cerning the intracellular proteolytic systems responsible for this accelerated protein breakdown\ has shown the implication of a non!lysosomal ATP!dependent pro! teolytic system which involves the conjugation of proteins that undergo degradation with ubiquitin "Argiles and Lopez!Soriano\ 0885#[ Interestingly\ Furuno et al[ "0889# studied the implication of the di}erent proteolytic path! ways in muscle during experimental denervation and con! cluded that\ although multiple proteolytic systems increase in parallel in the denervated muscle\ a non!lyso! somal process "independent of Ca1¦# appeared mainly responsible for the rapid loss of cell proteins\ especially of myo_brillar components[ On the same lines\ Medina et al[ "0884# have shown that the rise in the ATP!dependent  Corresponding author] Tel[] 23!82!3910991^ Fax] 23!82!3910448^ E!mail] argilesÝporthos[bio[ub[es

process correlates precisely with the overall enhancement of protein degradation and seems to account for most of the accelerated proteolysis following denervation atrophy in the rat[ Muscle protein degradation may represent an impor! tant factor in loss of body weight since skeletal muscle represents over 39) of total body mass in humans "Forbes\ 0876#[ Bearing this in mind\ a growing interest has developed concerning the proteolytic systems that are activated in skeletal muscle and that are responsible for muscle waste in pathological states[ The apparent selectivity of the ATP!ubiquitin!dependent pathway makes it an attractive mechanism to account for the speci_city of protein degradation within skeletal muscle\ and recent studies have dealt with the role of ubiquitin in muscle[ Although di}erent studies have focused on the activation of muscle proteolysis during experimental muscle atrophy models "Medina et al[\ 0880\ 0884#\ virtually no data concerning human pathologies is avail! able[ Bearing all this in mind\ the aim of the present inves! tigation was to test whether the ubiquitin!dependent pro! teolytic system is activated in human muscle showing neurogenic involvement "Authier et al[\ 0886# through changes in gene expression[

9086!9075:88:, ! see front matter Þ 0888 Elsevier Science Ltd[ All rights reserved[ PII] S 9 0 8 6 ! 9 0 7 5 " 8 7 # 9 9 9 7 9 ! 0

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1[ Experimental 1[0[ Human subjects and muscle biopsies The study included peroneus brevis muscle biopsy samples from 09 patients with neurogenic muscle involvement "his! tological changes due to skeletal muscle denervation#\ and normal deltoid muscle biopsy samples from _ve patients with normal muscle histology\ who underwent muscle biopsy to rule out a mitochondrial cytopathy[ Muscle sam! ples were conventionally processed for hematoxylin!eosin\ Masson trichrome\ ATPase "at pH 8[3\ 3[5 and 3[2#\ NADH!tetrazolium reductase\ succinate dehydrogenase and cytochrome C oxidase histoenzymatic reactions[ The criteria used to de_ne the di}erent neurogenic muscle _ber changes were those of Dubowitz et al[ "0874#[ Two sub! groups of patients were distinguished on the grounds of the most prominent histopathological _ndings ] "0# target _bers "TF#\ that were characterized by the presence of three concentric rings of di}erent stainings on NADH! tretrazolium reductase "NADH!TR# histoenzymatic reac! tion "29) of the _bers being at least a}ected#\ and "1# hyperoxidative _bers "HR#\ disseminated dark angulated _bers in NADH!TR staining "4 cases#[ Control muscle were normal muscle without histopathological abnor! malities[ The research received prior approval by the appropriate institutional review body and informed con! sent was obtained from each patient[ 1[1[ Immunochemistry Immunochemical detection of ubiquitin was performed on frozen sections\ using a polyclonal antibody against ubiquitin "Dako\ Glostrup\ Denmark# diluted 0:099[ Revelation of the staining was carried out using immu! noperoxidase assay "LSABþ1 kit\ Dako#[ To assess the speci_city of immunostaining\ the procedure was per! formed without primary antibody[ 1[2[ RNA isolation and Northern blot analysis Total RNA from muscle was extracted using the acid guanidinium isothiocyanate:phenol:chloroform method as described by Chomczynski and Sacchi "0876#[ RNA samples "19 mg:ml# were denaturated\ subject to elec! trophoresis in 0[1) agarose gels containing 5[2) for! maldehyde\ and transferred to Hybond N membranes "Amersham#[ RNA was _xed to membrane by illumi! nating with UV for 3 min[ The RNA in gels and in _lters was visualized with ethidium bromide and photographed by UV transillumination to ensure the integrity of RNA\ to check the loading of equivalents amounts of RNA\ and to con_rm proper transfer[ RNA was transferred in 19× standard saline citrate "SSC^ 9[04 M NaCl and 04 mM sodium citrate\ pH 6[9#[ Hydridization was done at 54>C overnight in the hybridization bu}er "9[14 M

Na1HPO3:6) SDS:0 mM EDTA:0) BSA:09) dextran sulphate#\ denatured labelled probes "095Ð096 cpm:ml# being added[ Radiolabelled probes were prepared by the random primer method "Boehringer#[ The ubiquitin probe used was a cDNA clone containing 01 pairs of the second ubiquitin coding sequence plus a complete third and fourth ubiquitin coding sequence and 019 base pairs of the 2?!untranslated region of the chicken polyubiquitin gene UBI "Bond and Schlesinger\ 0874#[ Filters were exposed to Hyper_lm!MP "Amersham# at −69>C for 1Ð3 days and the _lms quanti_ed by laser densitometry[ 1[3[ Biochemicals All enzymes and coenzymes were either obtained from Boehringer Mannheim\ S[A[ "Barcelona\ Spain# or from Sigma "St Louis\ MO\ U[S[A[#[ 1[4[ Statistical analysis Statistical analysis of the data was performed by means of the Student|s t!test[

2[ Results and discussion The intracellular degradation of proteins in eukaryotes is dependent on two basic pathways operating on two di}er! ent compartments[ One of them is located in the lysosomes and is involved in the degradation of endocytosed and membrane proteins "Dice\ 0876#[ This lysosomal pathway\ which basically involves the proteolytic enzymes known as cathepsines\ is relatively non!selective and is mainly responsible for basal degradation of long!lived proteins "Riley et al[\ 0877#[ In most mammalian cells\ protein breakdown increases up to twofold by this autophagic pathway upon deprivation of glucose or insulin "Dice\ 0876#[ The second pathway is cytosolic\ ATP!dependent and much more speci_c in recognizing signals on substra! tes[ This ATP!dependent mechanism involves the presence of a 7[5 kDa peptide known as ubiquitin[ Ubiquitin was _rst described in 0864 as a polypeptide with lymphocyte di}erentiation properties\ such was probably present in all cells "Goldstein et al[\ 0864#[ Later on\ a factor was ident! i_ed in reticulocyte lysates which stimulated the cytosolic ATP!dependent proteolysis[ Proteins were attached coval! ently to this factor\ thus becoming ubiquitinated and being then the target for proteolysis by cytosolic proteases "Hershko\ 0877#[ In addition\ proteins can be multi! ubiquitinated and this seems to be a requirement for e}ec! tive protein degradation in vivo[ Ubiquitin conjugates are degraded through the proteasome or multicatalytic pro! tease complex 15S "see Orlowsky "0889# and Argiles and Lopez!Soriano "0885# for review#[ Llovera et al[ "0883# have reported that both the pres! ence of ubiquitin conjugates and the expression of the

M[ Llovera et al[ : Neurochem[ Int[ 23 "0888# 026Ð039

di}erent ubiquitin genes is highly elevated in the skeletal muscle of cachectic tumour!bearing rats[ Other obser! vations have reported increases in the activity of the ATP!dependent proteolytic system in skeletal muscle of experimental animals during infection "Tiao et al[\ 0883^ Garc(a!Mart(nez et al[\ 0884#\ acidosis "Mitch et al[\ 0883#\ insulinopenia "Russ Price et al[\ 0885# and dener! vation atrophy "Medina et al[\ 0880\ 0884#\ all of these pathological states involving a considerable degree of

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muscle waste[ However\ a considerable lack of human data exists related to these pathological states[ On these lines\ we have recently reported an activation of the sys! tem in the muscle of slim AIDS patients "Llovera et al[\ 0887#[ We thought it would therefore be of major interest to see if the ubiquitin!dependent proteolytic system was also involved in human muscle[ Fig[ 0 shows the Northern blots for ubiquitin gene expression[ It can be seen that patients with muscle under!

Fig[ 0[ Expression of the genes corresponding to the ubiquitin!dependent proteolytic systems in human skeletal muscle[ Northern blots showing the expression of ubiquitin mRNAs in muscle extracts from control "C#\ target _bers "TF#\ and hyperoxidative _bers "HR#[ Target _bers were characterized by the presence of three concentric rings of di}erent stainings on NADH!tretrazolium reductase "NADH!TR# histoenzymatic reaction "29) of the _bers being at least a}ected# and HR were disseminated dark angulated hyperoxidative _bers in NADH!TR staining[ Control muscle was normal muscle tissue without histopathological abnormalities[ Transcript expression was detected after hybridization with a cDNA probe containing a region of the chicken polyubiquitin gene UBI[ Autoradiographs were subjected to scanning densitometry[ ethidium bromide "EtBr# was used for total RNA quantitation[ The results of _ve di}erent patients for each group are shown and expressed as arbitrary units as mean2S[E[M[ Statistical signi_cance of the di}erences]  P ³ 9[90[

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going neurogenic changes show an important increase in both the 1[3 kb "4[1! and 2[8!fold for target _bers "TF# and disseminated dark angulated hyperoxidative _bers "HF#\ respectively#\ and the 0[1 kb "2[8! and 3[3!fold for TF and HF\ respectively# gene transcripts[ The increase was statistically signi_cant in the case of the HF for the 1[3 kb transcript^ the rest of the values showed a marked increased tendency "in comparison with the controls# although the di}erences did not reach statistical sig! ni_cance[ It may be thus concluded that the ubiquitin system is involved in the muscle abnormalities observed in the patients[ In addition\ all the muscle samples of the patients tested in the present study showed a positive immunostaining for ubiquitin[ Interestingly\ TF show a considerable expression of the cytokine interleukin!0!a "IL!0a# while the HF does not show any expression of the cytokine "Authier et al[\ 0886#[ This observation suggests that the local production of the cytokine does not seem to be responsible for the increased gene expression and that other circulating factors "possibly other cytokines\ such as tumour necrosis factor!a "TNF## may have a role in this process in a similar fashion as to what is found in cancer cachexia "Costelli et al[\ 0882# and sepsis "Garc(a! Mart(nez et al[\ 0884#[ Present investigations are being carried out to elucidate this point[

Acknowledgements This work was supported by grants from the Fondo de Investigaciones Sanitarias de la Seguridad Social "F[I[S[# "86:1948# of the Spanish Health Ministry\ from the DGICYT "PB 83!9827# of the Spanish Ministry of Edu! cation and Science and from the Fundacio Pi i Sunyer "E99556#[

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Costelli\ P[\ Carbo\ N[\ Tessitore\ L[\ Bagby\ G[J[\ Lopez!Soriano\ F[J[\ Argiles\ J[M[\ Baccino\ F[M[\ 0882[ Tumor necrosis factor!a mediates changes in tissue protein turnover in a rat cancer cachexia model[ J[ Clin[ Invest[ 81\ 1672Ð1678[ Dice\ J[F[\ 0876[ Molecular determinants of protein half!lives in euka! riotic cells[ FASEB J[ 0\ 238Ð245[ Dubowitz\ V[\ Sewry\ C[A[\ Fitzsimons\ R[B[\ 0874[ De_nition of patho! logical changes seen in muscle biopsies[ In] Muscle Biopsy\ Bailliere! Tindall\ London\ pp[ 71Ð017[ Forbes\ G[B[\ 0876[ In] Human Body Composition] Growth\ Aging\ Nutrition and Activity[ Springer!Verlag\ New York\ p[ 060[ Furuno\ K[\ Goodman\ M[N[\ Goldberg\ A[L[\ 0889[ Role of di}erent proteolytic systems in the degradation of muscle proteins during denervation atrophy[ J[ Biol[ Chem[ 154\ 7449Ð7446[ Garc(a!Mart(nez\ C[\ Llovera\ M[\ Agell\ N[\ Lopez!Soriano\ F[J[\ Argiles\ J[M[\ 0884[ Ubiquitin gene expression in skeletal muscle is increased during sepsis[ Involvement of TNF!a but not IL!0[ Biochem[ Biophys[ Res[ Commun[ 106\ 728Ð733[ Goldstein\ G[\ Scheid\ M[\ Hammerling\ U[\ Boyse\ E[A[\ Schlesinger\ D[H[\ Niall\ H[D[\ 0864[ Isolation of a polypeptide that has lym! phocyte!di}erentiating properties and is probably represented uni! versally in living cells[ Proc[ Natl[ Acad[ Sci[ U[S[A[ 61\ 00Ð04[ Hershko\ A[\ 0877[ Ubiquitin mediated protein degradation[ J[ Biol[ Chem[ 152\ 04126Ð04139[ Llovera\ M[\ Garc(a!Mart(nez\ C[\ Agell\ N[\ Marzabal\ M[\ Lopez! Soriano\ F[J[\ Argiles\ J[M[\ 0883[ Ubiquitin gene expression is increased in skeletal muscle of tumour!bearing rats[ FEBS Lett[ 227\ 200Ð207[ Llovera\ M[\ Garc(a!Mart(nez\ C[\ Agell\ N[\ Lopez!Soriano\ F[J[\ Authier\ F[J[\ Gherardi\ R[K[\ Argiles\ J[M[\ 0887[ Ubiquitin and proteasome gene expression is increased in skeletal muscle of slim AIDS patients[ Int[ J[ Mol[ Med[ 1\ 58Ð62[ Medina\ R[\ Wing\ S[S[\ Goldberg\ A[L[\ 0880[ Activation of the ubiqui! tin!ATP!dependent proteolytic system in skeletal muscle during fasting and denervation atrophy[ Biomed[ Biochim[ Acta 49\ 236Ð 245[ Medina\ R[\ Wing\ S[S[\ Goldberg\ A[L[\ 0884[ Increase in levels of polyubiquitin and proteasome mRNA in skeletal muscle during starvation and denervation atrophy[ Biochem[ J[ 296\ 520Ð526[ Mitch\ W[E[\ Medina\ R[\ Grieber\ S[\ May\ R[C[\ England\ B[K[\ Price\ S[R[\ Bailey\ J[L[\ Goldberg\ A[L[\ 0883[ Metabolic acidosis stimulates muscle protein degradation by activating the adenosine triphosphate!dependent pathway involving ubiquitin and pro! teasomes[ J[ Clin[ Invest[ 82\ 1016Ð1022[ Orlowsky\ M[\ 0889[ The multicatalytic protease complex\ a major extralysosomal proteolytic system[ Biochemistry 18\ 09178Ð09186[ Riley\ D[A[\ Bain\ J[L[W[\ Ellis\ S[\ Haas\ A[L[\ 0877[ Quantitation and immunocytochemical localization of ubiquitin conjugates within rat red and white skeletal muscles[ J[ Histochem[ Cytochem[ 25\ 510Ð 521[ Russ Price\ S[\ Bailey\ J[L[\ Wang\ X[\ Jurkovitz\ C[\ England\ B[K[\ Ding\ X[\ Philips\ L[S[\ Mitch\ W[E[\ 0885[ Muscle wasting in insu! linopenic rats results from activation of the ATP!dependent\ ubiqui! tin!proteasome proteolytic pathway by a mechanism including gene transcription[ J[ Clin[ Invest[ 87\ 0692Ð0697[ Tiao\ G[\ Fagan\ J[M[\ Samuels\ N[\ James\ J[H[\ Hudson\ K[\ Lieb! erman\ M[\ Fischer\ J[E[\ Hasselgren\ P[O[\ 0883[ Sepsis stimulates non!lysosomal\ energy!dependent proteolysis and increases ubiqui! tin mRNA levels in rat skeletal muscle[ J[ Clin[ Invest[ 83\ 1144Ð 1153[