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by: Jaden Stiedemann


Jaden Stiedemann
Rice University
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Date Created: 10/19/15
1146 REVIEW Biodiversity Conservation and the Eradication of Poverty William M Adams1 Ros Aveling2 Dan Brockinglon3 Barney Dickson2 Jo Elliott l 2 Jon Hutton4 Dilys Roe5 Bhaskar Vira1 William Wo m conservation and poverty It is widely accepted that biodiversity loss and poverty are linked problems and that reduction should be tackled together However success with integrated strategies is elusive There is sharp debate about the social impacts of conservation programs and the success of communitybased approaches to conservation Clear conceptual frameworks are needed if policies in these two areas are to be combined We review the links between pove tion and present a conceptual typology of these relationships alleviation and biodiversity conserva r7 ination 21 22 There is a clear need for these to be integrated with national sustain able development strategies 1 23 The UN MDGs are premised on such integration th the area of land protected to maintain biological diversity being an indicator of performance against MDG Goal 7 to ensure quot39 How iodiversity conservation scientists B face a dilemma There is increasing concern that global efforts to maintain biodiversity are in conflict with those to reduce poverty 1 The decline of popula tions extinction of species and habitat century has been the creation of protected areas 3 Technical capacity to design effective protected area systems is increasing 4 allowing the identification of coverage and remaining gaps in the intema tional protected area system 5 This com bined with positive assessments of the effectiveness of protected areas is encourag ing the consolidation and expansion of the network of protected areas 6 The 2004 World Database on Protected Areas includes over 105000 sites covering an area of 197 million km 2 7 The establishment and effective management of a global series of protected areas was a key element of the 7th Conference of the Parties COP to the Convention on Biological Diversity CBD in 2004 8 The problem with this strategy is that its poverty are often negative The d use options with potentially signi cant economic opportunity costs 9 The creation of protected areas can 39Department of Geography University of Cambridge Cambridge CBZ 3EN UK 2Fauna amp Flora Interna tional Great Eastern House Tenison Road Cam bridge CB I ZRS UK 3School of Geography and the Environment University of Oxford Mans eld Road UK AResource Africa Post Office partment for International Development 1 Palace Street London W IE SHE UK 6International Institute for Environ ment and Development 3 Endsleigh Street London WC IH ODD UK Institute of Development Studies University of Sussex Falmer Sussex BN1 9RE UK have substantial negative impacts on local peo le The eviction of former occupiers or right holders in land or resources can cause the exacerbation of poverty as well as con travention of legal or human rights 10 14 Globally it is recognized that the costs of 39 39 rvation are not distributed cally many of the costs of protected areas in poor biodiverse countries are paid by local people 16 The 7th CBD COP called for an assessment of the economic and socio cultural costs of protected areas including the cost of livelihood opportunities foregone and policies to ensure that they are equitably compensated 8 By the start of the 21st m n g elimination had made the relation between biodiversity conservation and poverty reduc tion an important element of debate about conservation policy 1 13 e meaning of poverty may be intui tively obvious but its measurement is com ex Common definitions are based on monetary such as per capita income or nonmonetary such as health or mortality criteria although broader approaches have been suggested 17 18 In 1999 12 billion people worldwide had consumption levels below 1 a day and 28 billion lived on less than 2 a day 17 Poverty is not a static condition but it is estimated that between 300 and 420 million people live in a state of chronic poverty always or usually poor 19 The first of the United Nations Millen nium Development Goals MDGs agreed on in 2000 was to halve between 1990 and 2015 both the proportion of people whose income is less than 1 a day and the propor tion of people who suffer from hunger 20 National poverty reduction strategies are central to attempts to achieve poverty elim ever the co listing of poverty elimination d environmental goals does not mean that integrated solutions are possible or that protec ed areas can contribute to gro and poverty reduction in poor countries Indeed the separation by the MDGs of environmen tal sustainability issues from development oa s alarms some observers 24 It has even been suggested that the urgent global push for poverty reduction has subsumed or supplanted conservation goals 1 Combining Conservation and Development Goals The combination of poverty elimination and biodiversity conservation goals has been approached in various ways The specific problem of the social impacts of protected areas has been recognized by conservation planners for two decades The principle that the needs of local people should be system atically integrated into protected area plan ning was agreed to at the third World Parks Congress in Bali in 1982 25 In 1992 the president of IUCN The World Conserva tion Union argued that if local people do not support protected areas then protected areas cannot last 26 IUCN s director gener al now suggests that protected areas should 5 n m n n I m m with their bene ts extending far beyond their boundaries 27 but this is still an aspiration Delegates from the human rights and minority movements prominently voiced concern at the persistence of such impacts at the fifth World Parks Congress in September 2003 28 There are coherent calls for better understanding of the social impacts of protected areas 29 30 Beyond protected areas the question of whether it is possible to combine poverty elimination and biodiversity conservation relates to the more general debate familiar 12 NOVEMBER 2004 VOL to conservation scientists about the environ mental dimensions of development In the 20th century the dominant approach was to push for economic growth first and assume that environmental problems and indeed improved social welfare could be sorted out later Economists argue that as economies grow e can invest in cleaner technologies be observed in industrialized and newly industrialized countries with improvements in factors such as air pollution 31 In the 1950s and 1960s development planners paid scant attention to environmental impacts whether focusing on poverty elimination the creation of high productivity agriculture or physical infrastructure such as dams or industrialization and the associated problems of pollution 32 33 Critics of this technocratic top down de velopment focused on its environmental and vironmental aspects of development in the concept of sustainable development which underpinned the 1980 World Conservation Strategy document 33 As developed since notably at the World Conference on Environ ment and Development in Rio de Janeiro in 1992 and the World Summit on Sustainable Development in Johannesburg in 2002 the concept of sustainable development was extended to make explicit reference to justice equity and the elimination of poverty World leaders agreed that biodiversity and resource conservation must be fully integrated into strategies for economic development and are essential elements of sustainable livelihoods at local scales 35 It is widely argued that 39 39 39 erpins the livelihood strate gies of the rural poor 16 These political and policy insights have been accompanied by the emerg new academic subfields that offer integrative transdisciplinary insights into social ecological systems 36 Sceptics point to the large element of wish fulfillment in arguments about the pos sibility of win win solutions in sustainable development 1 33 37 38 A strong body of opinion however maintains that poverty elimination and conservation can ha together The term pro poor conservation has been used to identify conservation strategies that are designed to deliver both poverty reduction and biodiversity protection 39 40 But is this confidence in win win solutions justified Lasting positive out n d and poverty reduction simultaneously it may only be possible under specific institutional ecological and developmental conditions such as in long lasting eld projects in small human communities in fragile ecosys tems 1 The links between biodiversity and livelihoods and between conservation and p erty reduction are dynamic and locally specific 34 45 In most cases hard choices will be necessary between goals with signif icant costs to one goal or the other The acceptability of these costs will vary for different organizations and actors Diverse Relations Between Conservation and Poverty Reduction Clarity over the choices between biodiversity conservation and poverty elimination goals is essential The desire to package projects as delivering win win solutions plays down the incompatibilities between goals Equally exclusive conservation or development goals can be blind to alliances that favor both 1 We therefore offer a conceptual typology of the relationships between poverty reduction and conservation in order to promote a clearer understanding of them The typology presents four different ways of looking at the connections and disconnections between poverty reduction and conservation re ect ing positions in the current debate It includes 0 e moral and pragmatic dimensions of arguments for the conservation of biodiversity and the reduction of poverty Disentangling these makes for c ari 1 Poverty and conservation are separate policy realms This position sees poverty elimination and conservation as quite differ ent problems comprising distinct sectors of po i y c rn Thus conservation is a le itimate objective that can be pursued independently of any benefits in poverty reduction and vice versa Under this position conservation strategies would focus on the establishment of protected areas or approaches such as direct payments 46 If poverty is recognized as an important cause of conservation failure the response is the designation of further critical biodiverse habitat and the stronger defense of protected areas rather than the dissipation of scarce onservation resources to maintain diversi across landscapes or in poverty alleviation activities 37 38 This position sees conser vation benefiting poverty reduction indirect ly where it secures ecosystem services that yield economic benefits to society such as enhanced water yields from forested catch ments 47 48 There may also be local comes of w1th r projects are elusive 41 42 Projects that seek to integrate conservation and develop ment have tended to be overambitious and underachieving 41 44 Although it is de sirable to satisfy the goals of biodiversity wwwsciencemagorg SCIENCE VOL 306 for win win strategies that combine biodiversity and poverty reduction such as protected area tourism arrangements However this position holds that trying to combine conservation with poverty reduction everywhere risks misallocating REVIEW limited conservation resources and compro mising biodiversity preservation 37 38 The key to the success of conservation is the establishment and effective management of a complete global network of protected areas selected because of scientific criteria and owned or legally established by the state or legitimate private owners uccess is measured in terms of biodiversity criteria not of measures of social development 2 Poverty is a critical constraint on conservation This position makes the em pirical ra atic argument that poverty fail if it does not successfully address pov erty elimination Such a position might be expected in a scenario where poor people were overharvesting wild species poachin critical species or colonizing and cultivating biodiverse land and if the political or eco nomic costs of stopping them such as by a conventional strict protected area strategy were prohibitive Poverty reduction would be rtak n in this instance sim ly s means to achieve more effective conserva tion This position holds that to achieve its goal conservation must provide and be seen to provide effective contributions to poverty reduction including both net benefits to the o d e avoidance of significant local costs to any social group Conservation organizations will invest in addressing the poverty of critical protected area neighbors and actors with the power to disrupt conser vation programs Examples of policy action include classic park outreach strategies such as service provision to neighboring villages employment for local people and participa tion in park planning processes and income generating projects such as sharing revenue 0 wi l39 e tourism in protected areas integrated conservation development proj ects or the provision of locally acceptable alternatives to lost resources 41 43 3 Conservation should not compromise poverty reduction This position recognizes that conservation agencies have conservation as their primary goal but it holds that in pursuing that goal they should at a mini adopted at the Fifth World Parks Congress in 2003 but has its critics 27 Examples of strategies resulting from this position might include codes of conduct for conservation organizations social impact assessment of protected areas 29 30 and the payment of the full local opportunity costs of conserva tion in protected areas 50 Conservation strategies might also seek to generate posi tive economic benefits for local communities within constraints of biodiversity conserva tion targets for example through nonextrac tive use such as ecotourism 49 1148 REVIEW harvesting within sustainable limits such as safari hunting medicinal products or bio 9 z o E 2r 8 98 5 1 o V F is moral and political obligations on conserva tion agencies to take account of human pove t is a claim that recognizes that conservation action can be sustained despite negative social impacts 53 It applies even where it is possible to do conservation effectively without bene ting poor people 4 Poverty reduction depends on living resource conservation This position rests on the empirical claim that nancially poor and socially and politically marginalized people depend on living species in biodiverse eco systems for livelihoods and ecosystem ser vices d that their livelihoods can improved through appropriate conservation activities 33 Conservation is therefore a tool for achieving poverty reduction with the sustainable use of natural resources bein a foundation of strategies for ac ieving poverty reduction and social justice Biodi versity benefits not immediately necessary to this goal are a secondary gain This position might lead to the rejection ofa protected area strategy because except under special cir cumstances for example where shares 0 ecotourism revenues exceeded all other forms of land use protected areas were unlikely to achieve poverty reduction goals Alternative approaches would include the sustainable use of living resources to optimize economic r r positive impacts on the rur 1 within the constraints of ecological sustain ability such as fisheries wildlife grazing or forestry that are targeted at improving the livelihoods of the poor 54 56 Conservation in response to this position tends towar the maintenance of yields of harvestable species and ecosystems rather than the preservation of biodiversity Outcomes may deviate to a greater or lesser degree from biodiversity con servation targets This principle is re ected in the ecosystem approach adopted by the CBD in 2000 57 Conclusion No position outlined here suggests that either the conservation of biodiversity or the elim ination of poverty are improper goals All positions are consistent with the call for con servation organizations to identify and mon itor the social impacts of their work and to take corporate responsibility for operating in a socially accountable manner ey are also all consistent with the need for poverty alleviation efforts and wider projects for the development of humankind to have regard to their demands or footprint on the bio sphere 3 58 59 Different agencies and different individ uals are likely to wish to adopt different po sitions For example differences in thinking about the balance to be struck between pov erty reduction and biodiversity conservation underlie different positions in the par s ver sus sustainable use debate 3 7 38 54 60 Those advocating strictly enforced protected areas in poor developing countries to guaran tee the maintenance of populations of vul nerable species such as forest primates are adopting position 1 treating the problems of extinction and verty as separate Those advocating programs to tackle the poverty of people living around such parks in order to persuade them not to trespass or hunt are adopting position 2 seeing poverty as a crit ical constraint on conservation Those who would seek to increase the ow of revenues from such parks to a level that would fully compensate all stakeholders for associated op portunity costs of the park are adopting posi tion 3 attempting to ensure that conservation does not increase poverty in any way Those who propose conservation strategies building on the needs of local communities for sustain able harvests of wild species resources and not necessarily a formally declared protected area at all are adopting position 4 seeing conservation strategies based on sustainable use primarily as a means to reduce poverty olic that fails to take account of the diverse relationships between conservation eman s of poverty reduction and the related consumptive demands of the growing world economy risks failure 1 Organizations committe to the preservation of species and those committed to sustain able rural livelihoods based on natural re source use are likely to engage with issues of poverty and biodiversity in very different ways Their interactions will be facilitated if ey un erstand their mutual positions The recognition of different starting points in the way in which biodiversity conservation and poverty elimination goals are prioritized is essential if there is to be success in iden 39 ground and differences evelopment orga nizations Such recognition will facilitate the task of those who believe that the goals must be achieved together It is premature to abandon attempts to combine conservation and development The elimination of poverty an reservation of biodiversity are two distinct objectives Each may be driven by different moral agendas but there is considerable overlap in practice At the local scale the policy need is to reconcile the interests of different stake holders in the management of the natural resources ofbiodiverse ecosystems 45 The E larger challenge is to allow human society to meet its potential and share the fmits of economic gro while sustaining a biosphere that not only sustains full ecological functions but retains its living diversity 3 34 References and Notes 1 S E Sanderson K H Redford Oryx 37 1 2003 2 S Palumbi Science 293 W786 200 l W M Adams Against Extinction The Story of Conservation Earthscan London 2004 C R Margules R L Pressey Nature 405 243 2000 A S Rodrigues et al Nature 428 540 2004 A G Brunner R E Gullison R E Rice G A B da Fonseca Science 291 l25 200 httpseaunepwcmcorgwdbpa 5 July 2004 w o 39vorg 22 February 2004 M NortonG iffiths C Southey Ecol Econ 12 125 5quot own LDQON Equot 9 1995 10 D Brockington Fortress Conservation The Preserva tion of the Mkomazi Game Reserve Tanzania Currey Oxford 2002 ngler in Contested Nature Promoting F n ler C New York Press Albany Nv 2003 pp 25 40 12 M Colchester Salvaging Nature indigenous Peoples Protected Areas and Biodiversity Conservation World Rainforest Movement Montevideo 2002 13 K chimire M Pimbert Social Change and ConSer vation Earthscan London 1997 14 C Ceisler R de Sousa Public Adm 15 MWelsAmbio 21 237 1992 15 D Roe Elliott Oryx 38 137 2004 17 World Bank Poverty Net wwwworldbankorgpoverty en Development as Freedom Oxford Univ Press Oxford 2001 9 wwwchronicpovertyorgchronicpovertyreport 2004htm 7 July 2004 www evelopmentgoaisorg 10 March 2004 1 wwwworldbankorgpovertystrategiesindexhtm 19 une 2004 Dev 21 159 2001 22 Bojo R C Reddy Poverty Reduction Strategies and Bank Environment Department Paper 85 World Bank Washington DC 2002 23 wwwunorgesasustdevnatiinfonsdsmap2002htm 19 June 2004 24 D Roe in The Millennium Development Coats Hitting or Missing the Point iiED London 2003 the Target 55 70 25 A Phillips George Wright Forum 20 s 2002 25 Ramphal in Parks for Life Report of the Nth World Congress on National Parks and Protected Areas McNeely Ed itiCN cland Switzerland 1993 pp 55 55 27 A Steiner New Sci 130 21 2003 39ucnorgthemeswcpawpc2003englishoutputs recommendationshtm 10 March 2 29 D Brockington K Sc midtSoltau Oryx 33 140 2004 30 C Ceisler in Contested Nature Promoting intema tional Biodiversity Conservation with Social justice in the Twenty First Century S R Brechin P R Wiishusen C L Fortwangler P C West Eds State Univ of New York Press Albany Nv pp 217 229 31 World Bank World Development Report 1992 Bevel R F Dasmann P Milton P H F REVIEW mun N Leaderawllllams Oryx 37 21s 2003 pg M Samcnera w Whanda om 33 1A3 54 H A Franks uexNat emur Manage 1 2 2003 Carmen5mm Strategrex Are Farlmg m Wen Afnca AS 7 Ke Unlv nfCallfnmla Press Berkeley CA 1999 200 55 R 39 m N as p 1 r 9x 17132002 6 1 KczlelL Saunders Edy LIImg Off BIDdvermy furthe Pom1ucM Glanrt Swl zerlanrt 2002 A7 A Balmfnrd etaL Sauce 291 950 02 lmmg m mummy and Br memy lune m 40 D Rum Hutmm swam K cmum M Sammm A3 5 c Dally ELL Nature Sen759 Snaetal Depen a l emu exManagemem HED Lnndnm 2001 57 www blndlvnrgdeclslnnsdefaultaspxm c p70 m Natural Emsynem 151m Press Washa Pnlrcy Matter 12 52 2003 2 A1 D Hulma M w Manama Edy Afncan erdlrfe amp mglnm DC 1997 my 200A Livelrhnndx 7m Pmmrxe and Perimmune 0f Cum A9 5 c sllng Eml Emu 29 1 3 M L lmhnffetaL Nature 1129 370 200 mumy Carmen5mm Currey Oxfnr 0 50 A Balmfnrrt 7 wnmm Oryx 37 233 2003 59 M Wackemager w Ree OurEmlngrcaanmpnm AZ s MummbedzU lm D 11 237 1999 51 a M Campbelh M K 0mm Unmvermgthe Hrddm Reduang Human Impact an Ear New Snclety A3 c s a P Mesa n 21 23 10731995 HanemValuatmnMethndXin oodlandandFmen Publlsher Gabrlnla 15mm Brlllsh Calumbra Brandnm Penple and Park Unlang emmmaanmcam Lnndnm 2002 1996 11 la r Carpenten om 3 333 1999 60 P R Wllshusem s R Brechlm c L Furtwangen P c AA M was K Pmtected Area wnh anal Cnmmunme World 52 9 2 5 West Sac Nat Remurce 151 17 2002 D s w K aanK Washlnglnm DC 9 3 D Brncklngmm PnlchMatter 12 22 2003 Science Books et al HOME PAGE rn gt the latest book reviews gt extensive revrew archive gt topical books received lists gt buy books online wwwsclencemagorgbooks wwwsciencemagcrg SCIENCE VOL 305 12 NOVEMBER 2004 1149 19 Apr 2004 2125 1 1 AR AR2267CB20703Jex AR2267CB20703sgm LaTeX2e20020118 P1 GC AR EVIEWS IN ADVANCElO 1 146annurevce1bio20 12103135836 39 occur before nal 39 39 139 4 39 rim 2 v I E V J w Annu Rev Cell Dev Biol 2004 2061786 L1 doi 101146annurevcellbio20012103135836 7 9 Copyright 2004 by Annual Reviews All rights reserved 4 D I R First published online as a Review in Advance on April 21 2004 REGULATION OF MUSCLE MASS BY MYOSTATIN Se Iin Lee Johns Hopkins University School ofMedicine Department ofMolecnlar Biology and Genetics Baltimore Maryland 21205 emailsjleejhmiedn Key Words chalone TGFIS family satellite cell myoblast latency I Abstract Myostatin is a secreted protein that acts as a negative regulator of skeletal muscle mass During embryo genesis myostatin is expressed by cells in the myotome and in developing skeletal muscle and acts to regulate the nal number of muscle bers that are formed During adult life myostatin protein is produced by skeletal muscle circulates in the blood and acts to limit muscle ber growth The existence of circulating tissuespeci c growth inhibitors of this type was hypothesized over 40 years ago to explain how sizes of individual tissues are controlled Skeletal muscle appears to be the rst example of a tissue whose size is controlled by this type of regulatory mechanism and myostatin appears to be the rst example of the longsought chalone CONTENTS INTRODUCTION r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r 61 Identi cation of Myostatin and Phenotype of Myostatin De cient Animals 62 Biosynthesis of Myostatin and Regulation of Myostatin Latency r r r r r r r r r r r r r r 65 Myostatin Signaling Pathway r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r 71 Cellular and Tissue Responses to Myostatin Signalng 74 Clinical Applications r r 76 CONCLUSIONS r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r 78 INTRODUCTION 1 139 1 Despite the rapid advances in our of M H involved in regulating cell differentiation and tissue formation in vertebrates rela tively little is known about the control of tissue size Over 40 years ago Bullough 1962 1965 proposed that tissue size is controlled by the activities of negative growth regulators that he dubbed chalones According to this hypothesis individ ual tissues secrete distinct chalones which circulate throughout the body and act to inhibit the growth of the tissue producing the speci c chalone Despite inten sive efforts in the ensuing years to provide experimental support for this theory 0743463404111000611400 61 19 Apr 2004 2125 AR AR226 CB20 O3tex AR226 CB20 O3sgm LaTeX2e20020118 P1 GCE AR REVIEWS IN ADVANCE101146annurevcellbio20012103135836 62 LEE no molecules having the essential properties of a chalone for any tissue were identi ed and this theory to eXplain the control of tissue mass was essentially abandoned Recent work suggests that at least one tissue may in fact utilize this general type of regulatory mechanism to control tissue mass This tissue is skeletal muscle and the key mediator appears to be myostatin Identi cation of Myostatin and Phenotype of MyostatinDe cient Animals Myostatin previously called GDP 8 was originally identi ed in a screen for novel mammalian members of the transforming growth factor B TGF B superfamily of growth and differentiation factors McPherron et al 1997 The predicted myo statin protein sequence has all the hallmarks present in other family members including an N terminal signal sequence a dibasic proteolytic processing site and a C terminal domain following the processing site which contains nine cysteine residues with their characteristic spacing In this C terminal region the myostatin sequence shows signi cant homology to other family members and together with the highly related protein GDF ll McPherron et al 1997 Gamer et al 1999 Nakashima et al 1999 de nes a distinct subgroup within the larger superfamily Figure 1a goat MSTN ovine MSTN bovine MSTN porcine MSTN canine MSTN equine MSTN rabbit MSTN baboon MSTN macaque MSTN human MSTN murine MSTN 39 rat MSTN chicken MSTN E quail MSTN turkey MSTN duck MSTN goose MSTN pigeon MSTN striped bass MSTN white perch MSTN white bass MSTN shi drum MSTN sea bream MSTN tilapia MSTN fugu MSTN rl rainbow trout MSTN salmon MSTN brook trout MSTN catfish MSTN zebrafish MSTN r human GDF11 39 rat GDF11 murine GDF11 zebrafish GDF11 269 I I I I I I 25 20 15 10 5 0 Figure 1 Sequence comparisons of myostatin and GDFll proteins among various species 19 Apr 2004 21 25 AR AR226CB2003tex AR226CB2003sgm LaTeX2e200201 18 Pl GCE AR REVIEWS IN ADVANCE101 146annurevcellbio20012103 135836 MYOSTATIN 63 The expression pattern of myostatin suggested that it might play a role in regu lating muscle development or function McPherron et a1 1997 In mice myostatin is expressed in the myotome compartment of developing somites beginning at em bryonic day 95 and continues to be expressed in developing skeletal muscles throughout embryogenesis In adult tissues myostatin is expressed almost exclu sively in skeletal muscle although clearly detectable levels of myostatin RNA are also present in adipose tissue All skeletal muscles examined to date express at least some myostatin RNA although the expression levels vary from muscle to muscle The function of myostatin was elucidated by gene targeting studies in mice McPherron et a1 1997 Mice carrying a deletion of the portion of the gene encoding the Cterminal domain of myostatin were shown to have dramatic and widespread increases in skeletal muscle mass with individual muscles weighing about twice as much as those of wildtype mice Figure 261 Analysis of sections prepared from muscles of homozygous mutant mice showed that these increases in muscle mass result from a combination of increased number of muscle bers hy perplasia and increased ber size hypertrophy Every skeletal muscle examined appears to be affected by the mutation and both males and females are affected Figure 2 a Increased muscling in forelimbs of a myostatin knockout bottom panel compared with wild type mouse top panel reprinted from McPherron et a1 1997 b Belgian Blue bull showing the double muscling phenotype reprinted from McPherron amp Lee 1997 19 Apr 2004 2125 AR AR2267CB20703tex AR2267CB20703sgm LaTeX2e20020118 P1 GCE AR EVIEWS 1N ADVANCElO1146annurevcelbio20012103135836 64 LEE proportionately The increases in muscle mass in myostatin null mice are observed at an early age and continue to be maintained for the life of the animal NcPherron amp Lee 2002 Signi cantly heterozygous mice are also affected albeit to a lesser degree muscles of heterozygous mice weigh approximately 25 more than those of wildtype mice suggesting that the effect of myostatin is dose dependent The phenotype of mice lacking myostatin suggested that myostatin normally functions as a negative regulator of muscle growth and it was on this basis that myostatin was given its name NcPherron et al 1997 These ndings raised the possibility that blocking myostatin activity might have important applications for both human therapeutics and agriculture As a result the myostatin gene has been analyzed in a large number of different species Figure 1b and has been found to be extraordinarily well conserved McPherron amp Lee 1997 The predicted myostatin protein sequences inthe C terminal J 39 39 39 among human mice rat pigs chickens and turkeys Myostatinrelated sequences have also been identi ed in a number of different sh species NcPherron amp Lee 1997 Maccatrozzo et al 2001 Ostbye et al 2001 Rescan et al 2001 Roberts amp Goetz 2001 Rodgers amp Weber 2001 Rodgers et al 2001 Kocabas et al 2002 However the predicted protein sequences in sh are signi cantly diverged from those of other vertebrates and are actually almost as closely related to GDFll as they are to myostatin These sequence comparisons taken together with the observation that these genes are expressed in a much wider range of tissues in sh have led to the suggestion that the divergence of myostatin and GDFll occurred after the origin of sh and that the functions of myostatin and GDFll in sh are carried out by genes representing the original ancestral precursor Ostbye et al 2001 Although no other putative sh GDFll orthologs have been published to date the deposition into the public databases by lliev Roberts and Goetz of a partial second zebra sh sequence accession number AF411599 much more closely related to GDFll than to myostatin suggests that the evolution of myostatin sequences and functions in sh may be more complicated Conservation of the myostatin sequence across species suggests conservation of function and in fact mutations in the myostatin gene have been shown to be responsible for the double muscling phenotype in cattle Figure 2b Grobet et al 1997 1998 Kambadur et al 1997 McPherron amp Lee 1997 For almost 200 years certain breeds of cattle have been recognized by breeders as being signi cantly more muscled than standard breeds Nenissier 1982 Genetic analysis of one of these socalled doublemuscled breeds Belgian Blue revealed that this trait segregated as a single locus on bovine chromosome 2 with a semidominant mode of inheritance Hanset amp Michaux 19 85 Charlier et al 1995 The increased muscle mass observed in myostatin null mice NcPherron et al 1997 taken together with mapping studies showing that the human myostatin gene is located in a region of human chromosome 2 syntenic to the portion of bovine chromosome 2 containing the double muscling locus Grobet et al 1997 McPherron amp Lee 1997 made the myostatin gene an obvious candidate for the double muscling locus in cattle lndeed analysis of 39 gene in a ofquot quotb1 mquot 1 A breed 19 Apr 2004 21 25 AR AR226CB2003tex AR226CB2003sgm LaTeX2e200201 18 P1 GCE AR REVIEWS IN ADVANCE101 146annurevcellbio20012103 135836 MYOSTATIN 65 has identi ed at least seven different mutations including premature stop codons frameshift mutations and point mutations at highly conserved amino acid residues Grobet et al 1997 1998 Kambadur et al 1997 McPherron amp Lee 1997 Cappucio et al 1998 Marchitelli et al 2003 Collectively these mutations account for the vast majority of doublemuscled breeds that have been characterized suggesting that the myostatin gene is the predominant if not sole locus for double muscling in cattle The identi cation of myostatin as a negative regulator of muscle growth has raised many important questions about the control of muscle growth Understand ing the mechanism by which myostatin regulates muscle mass will be critical not only for understanding the control of tissue size in general but also for developing new strategies for increasing muscle growth both for human therapeutic applica tions and for livestock production In the remainder of this review I describe the current state of knowledge of the regulatory mechanisms by which myostatin ac tivity is regulated the components of the myostatin signaling pathway the cellular and tissue responses to myostatin signaling and the potential ef cacy of blocking myostatin activity for clinical applications Biosynthesis of Myostatin and Regulation of Myostatin Latency Similar to other TGFB family members myostatin is synthesized as a precursor protein that undergoes two proteolytic processing events in order to generate the bi ologically active molecule Figure 3a McPherron et al 1997 The rst cleavage 3 I I E 9 Q Figure 3 Processing of myostatin protein Myostatin is synthesized in a precursor form that undergoes two proteolytic processing events one removes the N terminal signal sequence a second generates the C terminal fragment which possesses receptor binding activity a Following proteolytic processing the propeptide blue and the disul de linked C terminal dimer yellow remain bound non covalently in a latent complex b Activation of latent myostatin can occur by proteolytic cleavage of the propeptide b C by members of the BMP 1tolloid family of metalloproteinases which causes dissociation of the latent complex cl 19 Apr 2004 2125 AR AR2267CB20703tex AR2267CB20703sgm LaTeX2e20020118 P1 GCE AR EVIEWS 1N ADVANCElO l l46annurevcelbio20012103135836 66 LEE event removes the 24amino acid signal peptide necessary for targeting the protein to the secretory pathway The second cleavage event occurs at an RSRR Arg SerArgArg site at amino acids 2407243 numbered from the rst amino acid following the signal sequence generating Nterminal and Cterminal fragments with predicted nonglycosylated molecular masses of 27680 and 12400 respec tively The precise proteinase responsible for generating this second cleavage in vivo is not known T quot 39 394 preceding quot suggests that this cleavage is carried out by paired dibasic amino acidcleaving en yme PACE also called furin which is a member of the family of subtilisinlike serine proteinases Wise et al 1990 in fact a soluble form of PACE Rehemtulla amp Kaufman 1992 is capable of fully cleaving the myostatin precursor protein at the RSRR site when expressed in Chinese hamster ovary CHO cells Lee amp McPherron 2001 Thies et al 2001 Studies with C2C 12 cells have also suggested a possible role for metalloproteinases in processing of myostatin as treatment of C2C12 cells with small molecule inhibitors of metalloproteinases has been shown to result in production of an increased proportion of unprocessed myostatin as well as hypertrophy of myo bers presumably as a result of reduced levels of mature myostatin protein being produced by these cells Huet et al 2001 However no metalloproteinase has been identi ed to date that is capable of directly cleaving the myostatin precursor protein at the dibasic site The Cterminal fragment following proteolytic processing is the biologically active species and except when explicitly stated otherwise all subsequent refer ences to myostatin in this review should be taken to mean the C terminal dimer Al though the threedimensional structure of the Cterminal fragment has not yet been determined the high degree of sequence similarity between myostatin and other TGF family members suggests that the active form of myostatin is most likely a disul delinked dimer of C terminal fragments folded into a cystine knot structure as has been described for other family members Daopin et al 1992 Schlunegger amp Grutter1992 Grif th et al 1996 Hinck et al 1996 Mittletal1996 Eigenbrot amp Gerber 1997 Scheu er et al 1999 Kirsch et al 2000 Thompson et al 2003 Indeed myostatin produced by CHO cells can readily form ho modimers and as discussed below the puri ed homodimer is active in a va riety of in vitro assays including receptor binding and activation of reporter genes Lee amp McPherron 2001 Thies et al 2001 Rebbapragada et al 2003 Whether yostatin form quot WlL IJ utth TGF p iamily 39 known The Nterminal fragment following proteolytic processing has been most com monly referred to as the propeptide On the basis of what is known about other TGF family members the propeptide is presumed to play an important role in the proper folding of the C terminal domain into a cystine knot structure Various reports have described the puri cation of biologically active myostatin protein produced as an isolated C terminal fragment in bacteria for example see Thomas et al 2000 Taylor et al 2001 however the concentrations of bacterially pro duced myostatin protein required to demonstrate biological effects in these studies 19 Apr 2004 2125 AR AR2267CB20703 ex AR2267CB20703sgm LaTeX2e20020118 P1 GCE AR EVIEWS 1N ADVANCElO1146annurevcelbio20012103135836 MYOSTATIN 67 were quite high 1007500 MM suggesting that only a fraction of the puri ed preparation represented properly folded Cterminal dimers In addition to its role in proper folding of the precursor protein the propep tide also plays an important role in regulating the activity of the Cterminal dimer following proteolytic processing This regulatory role of the propeptide was rst elucidated from the analysis of CHO cells engineered to overproduce myostatin These cells were shown to secrete myostatin protein as a noncovalent complex of the propeptide with the Cterminal dimer Lee amp McPherron 2001 Figure 3b In this complex the propeptide maintains the Cterminal dimer in an inactive latent state Lee amp McPherron 2001 Thies et al 2001 Wolfman et al 2003 similar to that described for TGF Miyazono et al 1988 and as in the case of TGF this latent complex can be activated arti cially in vitro by treatment for example with heat Wolfman et al 2003 The ability of the propeptide to inhibit the biolog ical activity of the myostatin Cterminal dimer has been documented extensively in vitro and in vivo Speci cally the puri ed propeptide can block the activity of the puri ed myostatin Cterminal dimer in both receptor binding and reporter gene assays Lee amp McPherron 2001 Thies et al 2001 and transgenic mice overex pressing the propeptide in muscle phenocopy myostatin knockout mice in terms of increased muscle mass Lee amp McPherron 2001 Yang et al 2001 lthough the existence of the latent complex was originally elucidated by stud ies with CHO cells engineered to overproduce myostatin it is clear that this latent complex also exists in vivo In particular myostatin has been shown to circulate in the blood in a latent form that can be activated by acid treatment Zimmers et al 2002 The precise nature of this latent form is unknown but biochemical analy ses of proteins bound to myostatin have shown that at least one protein normally complexed to myostatin in the blood is the propeptide Hill et al 2002 consis tent with a normal role for the propeptide in regulating the activity of myostatin in vivo Therefore understanding how the propeptidemyostatin latent complex is activated should provide important insights into the mechanisms by which myo statin activity may be modulated in different physiological states In this respect one mechanism for activating myostatin latency appears to be proteolytic cleavage of the propeptide Wolfman et al 2003 Members of the bone morphogenetic protein BMP ltolloid family of metalloproteinases are capable of cleaving the propeptide immediately N terminal to aspartate 76 Figure 3bc All four proteinases in this family can cleave not only the puri ed propeptide but also the propeptide bound to the C terminal dimer and cleavage of the propeptide in this complex results in activation of the C terminal dimer The importance 0 these proteinases in regulating myostatin latency in vivo is supported by studies using a mutant form of the propeptide in which aspartate 76 was mutated to alanine This mutation renders the propeptide resistant to proteolysis by the BMP ltolloid proteinases in vitro and weekly injection of the mutant propeptide into mice over a span of four weeks resulted in increases in muscle growth of approximately 25 in contrast no effects on muscle mass were observed in similar experiments using wildtype propeptide The simplest interpretation of these results is that the 19 Apr 2004 2125 AR AR2267CB20703tex AR2267CB20703sgm LaTeX2e20020118 P1 GCE AR EVlEWS 1N ADVANCElO l l46annurevcelbio20012103135836 68 LEE mutant propeptide is capable of forming latent complexes with myostatin that are resistant to activation by proteinases in the BMPltolloid family This group of proteinases has previously been demonstrated to play an analogous role in regulating the activities of certain other TGF family members In particular members of the BMP ltolloid family are responsible for cleaving chordin which normally binds to and inhibits the activity of BMPs Blader et al 1997 Piccolo et al 1997 Marques et al 1997 Scott et al 1999 Pappano et al 2003 It is also interesting to note that one member of the BMPltolloid family TLL2 has been shown to be expressed speci cally in skeletal muscle during embryogenesis Scott et al 1999 raising the possibility that TLL2 may be the proteinase that activates myostatin during muscle development In addition to the propeptide several other proteins have also been shown to capable of binding and inhibiting the activity of the myostatin C terminal dimer One of these is follistatin which can bind to a number of other TGF family mem bers as well Nakamura et al 1990 Yamashita et al 1995 de Winter et al 1996 Fainsod et al 1997 lemura et al 1998 Several studies suggest that follistatin can function as a potent myostatin antagonist and plays an important role in modulat ing myostatin activity in vivo First follistatin is capable of blocking myostatin activity in both receptor binding and reporter gene assays Lee amp McPherron 2001 Zimmers et al 2002 as well as in nude mice implanted with myostatinexpressing cells Zimmers et al 2002 Second studies with chick embryos have shown that follistatin is expressed in myotomal cells and developing muscle at stages when myostatin is also expressed Amthor et al 1996 2002ab and that implantation of beads soaked with follistatin into developing wing buds can cause an upregulation of Fax3 expression and delay muscle cell differentiation Amthor et al 2002a Although these latter studies were interpreted in the context of BMP signaling these effects would also be consistent with follistatin antagonism of myostatin activity Finally genetic studies in mice have shown that overexpression of follis tatin in muscle can cause dramatic increases in muscle growth Lee amp McPherron 2001 and conversely that loss of follistatin results in reduced muscle mass at birth Natzuk et al 1995 which is what one might expect for excess myostatin signaling during embryonic development Follistatin also appears to play a role in regulating the activity of GDFll which is highly related to myostatin McPherron et al 1997 Gamer et al 1999 Nakashima et al 1999 As in the case of myostatin follistatin can act as a GDP 11 antagonist in vitro because follistatin is able to block GDFll activity in the Xenopus animal cap assay Gamer et al 1999 Moreover in addition to having reduced muscle mass at birth follistatin knockout mice have posteriorly directed homeotic transformations of the axial skeleton Natzuk et al 1995 This aspect of the follistatin knockout phenotype is noteworthy because Gdf Z knockout mice have extensive anteriorly directed homeotic transformations throughout the axial skeleton McPherron et al 1999 The contrasting phenotypes seen in Gde and follistatin knockout mice are consistent with the hypothesis that follistatin may normally be involved in antagonizing GDFll signaling in vivo 19 Apr 2004 2125 AR AR226 CB20 03teX AR226 CB20 03sgm LaTeX2e20020118 P1 GCE AR REVIEWS IN ADVANCE101146annurevcellbio20012103135836 MYOSTATIN 69 In addition to propeptide and follistatin two other proteins FLRG and GASP 1 also appear to be involved in regulating the activity of the myostatin C terminal dimer extracellularly Hill et al 2002 2003 Both FLRG and GASP l are com plexed to myostatin in the blood of mice and humans and studies with recombinant proteins have shown that both proteins can bind with high af nity to the myostatin C terminal dimer and inhibit its activity as assessed by reporter gene assays In terestingly GASP l can also bind directly to the propeptide in the absence of the C terminal dimer FLRG was originally identi ed as a follistatin related gene present at a chromosomal translocation breakpoint in a patient with B cell chronic lymphocytic leukemia and was shown to be capable of blocking the activity of the TGF family members activin and BMP 2 in in vitro assays Hayette et al 1998 Tsuchida et al 2000 GASP l is a novel protein that contains a follistatin related domain but in addition contains multiple domains found in proteinase inhibitors To date there is are no genetic data addressing the biological functions of these molecules in vivo The eXistence of multiple proteins capable of binding myostatin and inhibiting its activity Figure 4 raises many questions as to the speci c role that each protein plays in regulating myostatin activity It seems likely that the propeptide becomes bound to the myostatin C terminal dimer during the biosynthesis of the mature protein and remains bound following release of the protein into the circulation Activation of the C terminal dimer from this latent compleX presumably requires dissociation of the propeptide and proteolytic cleavage of the propeptide by BMP 1tolloid proteinases may be one mechanism for achieving activation What roles FLRG and GASP l play in regulating myostatin are less clear One possibility is that these proteins together with the propeptide may form ternary or higher order complexes with the myostatin C terminal dimer and that each of these proteins has a unique regulatory role in this compleX in maintaining myostatin latency In this GASP l 1 muscle BMP lTLD propeptide 1 myostatin growth T follistatin FLRG Figure 4 Myostatin activity is inhibited by extracellular binding proteins including its propeptide follistatin FLRG and GASPl The propeptide can be inactivated by proteolytic cleavage by BMPltolloid metalloproteinases 19 Apr 2004 2125 AR AR2267CB20703Jex AR2267CB20703sgm LaTeX2e20020118 P1 GCE AR EVIEWS lN ADVANCElO l l46annurevcelbio20012103135836 70 LEE respect GASPl can interact directly with both the propeptide and the C terminal dimer The possibility that GASPl may be able to form a ternary complex with the propeptide and C terminal dimer taken together with the observation that GASP 1 contains multiple proteinase inhibitory domains has led to the suggestion that GAS P l might be involved in regulating the activity of a proteinase that cleaves the propeptide to activate latency Hill et al 2003 An alternative possibility raised by these authors is that the role of GASPl is to regulate the activity of the proteinase which is involved in processing the myostatin precursor protein at the RSRR site however this role would be relevant only in terms of regulating the latent complex if cleavage of the precursor protein can normally occur either in the blood or at the myostatin target site A second possibility regarding 1 39 1 by LllC v u39 binding proteins is that the binding of the propeptide FLRG and GASPl t the myostatin Cterminal dimer may be mutually exclusive and as a result the circulating form of myostatin may actually be a heterogenous mixture of these various complexes Assuming that the initial complex formed is between the C terminal dimer and the propeptide immediately following proteolytic processing of p protein the 1 1 is that a 1 1 39 39 for binding of FLRG or GASPl to the C terminal dimer in this scenario would be dissociation of the propeptide from the C terminal dimer If multiple distinct latent complexes do exist in vivo an appealing notion is that the mechanisms involved in activating each of these complexes are distinct and therefore that each complex potentially is responsible for responding to different physiological stimuli to regulate the overall level of active myostatin protein A third possibility as suggested by Hill et al 2002 in the context of FLRG is that FLRG andor GAS P l may be involved in terminating myostatin signaling after myostatin has already bound to and activated its receptors According to this hypothesis these proteins could bind the myostatinreceptor complex and given that FLRG and GAS P l are bound to myostatin in the blood it is possible that these proteins can dissociate myostatin from its receptor and remain bound to myostatin following dissociation Because the complexes of myostatin with FLRG or GASP 1 could represent end products of the signaling pathway a reasonable prediction in this scenerio is that these complexes are incapable of being activated learly identifying the hue roles of each of these proteins requires not only additional biochemical approaches to further characterize the latent complexes but also genetic experiments in which the functions of FLRG and GASPl are elim inated Whatever the speci c roles of each of these regulatory proteins the fact that myostatin normally exists in latent complexes provides another level of regu latory possibilities beyond simply regulating expression of the myostatin mRNA and protein For this reason levels of myostatin mRNA and protein under vari ous physiological conditions or in response to various stimuli may not accurately re ect the actual level of myostatin signaling Understanding how myostatin is activated from this latent state will be critical not only for knowing how myostatin activity is regulated but also for identifying new strategies for the development of therapeutic agents for clinical applications 1 1 19 Apr 2004 2125 AR AR2267CB20703Jex AR2267CB20703sgm LaTeX2e20020118 P1 GCE AR EVIEWS 1N ADVANCElO1146annurevcelbio20012103135836 MYOSTATIN 71 Myostatin Signaling Pathway Upon activation from its latent state the myostatin C terminal dimer is capable of binding to receptors and activating a signal transduction cascade in the target cell There is now considerable evidence that myostatin signals by a mechanism similar to that of other TGF related ligands Most members of the TGF superfamily are believed to signal through heteromeric complexes of type 1 and type 11 ser inethreonine kinase receptors for review see Massagu 1998 Most ligands bind rst to a type II receptor and this ligandtype II receptor complex then recruits a type 1 receptor The type II receptor kinase then phosphorylates and activates the type 1 receptor kinase which in turn phosphorylates the Smad proteins The activated Smad proteins function as the key intracellular mediators of signaling by entering the nucleus to regulate expression of downstream genes Cr quot 39 quot 39 puiim i 39 mvo LatinproteillL strated that myostatin is cap ble of binding the activin type 11 receptors ActRlIA and ActRllB in vitro Lee amp McPherron 2001 Rebbapragada et al 2003 Qual itatively myostatin appears to bind more strongly to ActRllB than to ActRlIA however actual af nity determinations have been reported only for myostatin bind ing to ActRllB Lee amp McPherron 2001 The role of the activin type 11 receptors in mediating myostatin signaling in vivo is supported by transgenic mouse studies in which a dominantnegative form of ActRllB which retains the membrane span ning domain but lacks the kinase domain was expressed in muscle using a myosin light chain promoter and enhancer Lee amp McPherron 2001 These transgenic mice were shown to have dramatic increases in muscle mass comparable to those seen in myostatin knockout mice and as in the case of myostatin knockout mice the increase in muscle mass in these transgenic mice was shown to result from a combination of muscle ber hyperplasia and hypertrophy Although the phenotype of these mice is certainly consistent with a role for activin type 11 receptors in the myostatin signaling pathway one caveat to the interpretation of these studies is that the precise mechanism by which the dominantnegative ActRllB enhanced muscle growth in these mice is unclear It is possible for example that rather than blocking actual myostatin signaling in the target cell the truncated receptors may have acted competitively to bind free myostatin ligand and thereby prevent myostatin from binding to its true receptors More de nitive genetic support for a role for ActRlIA and ActRllB in mediating myostatin signaling will await the phenotypic analysis of the muscles of mice lacking one or both of these genes For GDFl l both biochemical and compelling genetic evidence support a role for activin type 11 receptors in mediating signaling Co immunoprecipitation stud ies carried out with Xenopus embryos have shown that similar to myostatin GDP 11 is capable of binding both ActRllB and ActRlIA and as in the case of myo statin GDFll appears to bind more strongly to ActRllB than to ActRlIA Oh et al 2002 Moreover the phenotype of Gde knockout mice McPherron et al 1999 is remarkably similar to that of mice de cient in activin type II receptor signaling Oh amp Li 1997 Oh et al 2002 As described above GdeZ knockout mice have extensive anteriorly directed transformations of the axial skeleton with 4 19 Apr 2004 2125 AR AR2267CB20703tex AR2267CB20703sgm LaTeX2e20020118 P1 GCE AR EVIEWS 1N ADVANCElO l l46annurevcelbio20012103135836 72 LEE the most dramatic manifestations being the presence of ve to six additional tho racic segments and two to three additional lumbar segments McPherron et al 1999 Likewise genetic studies in mice have shown that both ActRlIA and Ac tRllB also play important roles in regulating axial patterning Speci callyActRIIB knockout mice have anteriorly directed transformations including the presence of three additional thoracic segments Oh amp Li 1997 and although axial patterning appears normal in ActRIIA mice animals lacking both copies of ActRIIB and one copy of ActRIIA have a more severe phenotype than ActRIIB mice with most ActRIIB ActRIIA mice having four additional thoracic segments and one additional lumbar segment Oh et al 2002 The presumption is that mice lacking both copies of both ActRIIA and ActRIIB would have an even more se vere patterning defect although tissuespeci c or conditional knockouts will be required to address this point as the complete double knockout of these genes has been shown to result in early embryonic lethality Song et al 1999 This same general pattern has also been observed with respect to kidney development Gdf Z knockout mice have severe kidney defects with most mutant mice having com plete bilateral renal agenesis NcPherron et al 1999 Esquela amp Lee 2003 About 25 of ActRIIB mutant mice also have kidney defects Oh amp Li 1997 and this percentage is increased to about 98 in mice that also lack one copy of ActRIIA Oh et al 2002 Taken together these data provide strong support for the model that both ActRlIA and ActRHB are responsible for transducing the GDF 11 signal in vivo with ActRllB perhaps playing a more dominant role On the basis of the high degree of sequence relatedness between myostatin and GDFll and the ability of myostatin to bind activin type 11 receptors in vitro it seems likely that ActRlIA and ActRllB are also the relevant type 11 receptors for myostatin in vivo Myostatin and GDFll are the newest members of a growing list of ligands that appear to be capable of signaling through activin type 11 receptors Given that these receptors as well as many of these ligands have relatively widespread expression patterns and given that some of these ligands including myostatin itself circu late systemically a key question is how speci city of signaling is achieved For myostatin one possible mechanism for achieving speci city might be selective activation of the latent complex at the target site see above An attractive hypoth esis for example is that the BMP ltolloid proteinases responsible for cleaving the myostatin propeptide are activated only at sites and times at which suppression of muscle growth is desired Whether this type of regulatory mechanism operates in vivo will require a much more detailed understanding of how activation of latent myostatin is regulated under various physiological conditions An alternative mechanism for achieving speci city is selective utilization of coreceptors Certain members of the TGF family require one of several unrelated coreceptors in order to engage the type II receptor and it is noteworthy that in nearly every case in which this regulatory mechanism has been described to date it is the interaction of a ligand speci cally with the activin type 11 receptors that is modulated by the presence of these coreceptors In particular although the activins and BMPs are capable of binding activin type 11 receptors in the absence 19 Apr 2004 2125 AR AR2267CB20703Jex AR2267CB20703sgm LaTeX2e20020118 P1 GCE AR EVlEWS lN ADVANCElO l l46annurevcelbio20012103135836 MYOSTATIN 73 of coreceptors binding of inhibin requires the presence of betaglycan Lewis et al 2000 and binding of nodal and GDFl requires the presence of the EGFCFC proteins cripto and cryptic Schier amp Shen 2000 Cheng et al 2003 Whether a coreceptor is required for binding of myostatin to ActRlIA and ActRHB is not known However the receptor binding af nity of myostatin that has been determined for COS1 cells transfected with ActRllB is somewhat lower than typical af nities reported for other TGF family members and their cognate type II receptors Lee amp McPherron 2001 which raises the possibility that a receptor component for myostatin such as a coreceptor may have been limiting in COS1 cells The coreceptors are a group of unrelated ligands that somehow are involved in presenting ligands to the type II receptor Only certain ligands require the presence of a coreceptor The type 1 receptors actually signaling receptors are activated by ligand binding to the type II receptor All ligands require the activation of a type I receptor for signaling to take place A third possible mechanism for achieving speci city of signaling is restricted expression of the appropriate type 1 receptors that is only those cells express ing both the activin type II receptors and the appropriate type 1 receptors would be capable of transducing the myostatin signal In this regard there is evidence that certain members of the TGF superfamily can enhance the af nity of the ligand for the type II receptor Attisano et al 1993 Crosslinking studies with cells cotransfected with ActRllB and individual type 1 receptors have shown that myostatin can bind two type 1 receptors ALK4 and ALK5 Rebbapragada et al 2003 Similarly although a systematic survey of type 1 receptors has not yet been carried out for GDFl l studies using Xenopus embryos have shown that GDFll also can bind ALK4 Oh et al 2002 The interaction of ALK4 with activin type II receptors had been documented extensively in prior studies with other ligands for review see Massague 1998 However ALK5 had not previously been shown to be capable of interacting with type II receptors other than TGF RH Hence if ALK5 is involved in mediating myostatin andor GDFll signaling the unique combination of ALK5 with activin type II receptors certainly provides one possi ble mechanism for achieving speci city of signaling by these ligands In contrast to the activin type II receptors however there is are no genetic data as yet supporting a role for either ALK4 or ALK5 in mediating myostatin or GDFll signaling in vivo Whether ALK4 andor ALK5 are the signaling type 1 receptors for myostatin in vivo a variety of data suggests that myostatin signaling leads to activation of Smad proteins Treatment of cells in culture with puri ed myostatin protein has levels ofbothp p Q39 d 1 1 aswell a t39 t39 f quot 3 r 39 r LCl genes Thies et al 2001 Langley et al 2002 Rebbapragada et al 2003 Although less is known about the GDFll signaling pathway injection of Xenopus embryos with GDFll RNA also leads to increased levels of phosphoSmad2 Finally there is considerable genetic data showing that the nuclear protein cski which is capable of interacting with and blocking the activity of Smad 2 3 and 4 Luo et al 1999 Stroschein et al 19 Apr 2004 2125 AR AR2267CB20703tex AR2267CB20703sgm LaTeX2e20020118 P1 GCE AR EVIEWS 1N ADVANCElO l l46annurevcelbio20012103135836 74 LEE 1999 Sun et al 1999ab Akiyoshi et al 1999 is a potent regulator of muscle growth Mice lacking cski have a severe reduction in skeletal muscle mass Berk et al 1997 and transgenic mice overexpressing cski in muscle have dramatic muscle hypertrophy Sutrave et al 1990 The simplest interpretation of these data is that cski normally functions to block myostatin signaling in vivo by blocking activities of Smad proteins normally activated by myostatin Cellular and Tissue Responses to Myostatin Signaling Although relatively little is known about the downstream genes that are regulated 39 391 Qmad Protein ioni r ant WNW3 119 been made in terms Of iden tifying at least some of the cellular responses to myostatin signaling As discussed above mice carrying a deletion of myostatin have increases in both in the number of muscle bers and in ber sizes McPherron et al 1997 These ndings suggest that myostatin may play two distinct regulatory roles one to regulate the nal number of muscle bers during development and a second to regulate muscle ber growth postnatally The hypothesis that myostatin may have this dual role has been borne out by a variety of experiments showing that the development of hyperplasia versus hypertrophy depends on the method used to block myostatin activity For example similar to myostatin knockout mice transgenic mice expressing various myostatin inhibitors such as the propeptide follistatin or a dominantnegative form of ActRllB under the control of a myosin light chain promoterenhancer have increases in both ber numbers and ber sizes Lee amp McPherron 2001 contrast hyperplasia without hypertrophy has been reported in transgenic mice in which a chicken actin promoter coupled to a CMV enhancer was used to drive expression of a mutant form of myostatin in which an invariant cysteine residue in the Cterminal domain was changed to tyrosine N ishi et al 2002 Conversely transgenic mice in which a muscle creatine kinase promoter was used to express a mutant form of myostatin with an altered furin cleavage site have been shown to have hypertrophy without hyperplasia Zhu et al 2000 Similarly neutralizing an tibodies to myostatin are capable of causing signi cant increases in muscle growth when administered to adult mice and this increase in muscle growth appears to result entirely from an increase in muscle ber sizes Bogdanovich et al 2002 ittemore et al 2003 Taken together these results demonstrate that the effects of myostatin on ber numbers can be dissociated from its effects on ber sizes and that the development of hyperplasia versus hypertrophy is likely to be dependent on the time during embryonic development or postnatal life at which myostatin activity is lost The regulation of ber numbers by myostatin most likely results from direct effects of myostatin on proliferation andor differentiation of myoblasts during development A number of studies have examined the effects of myostatin on C2C12 myoblasts either by treating cells with puri ed recombinant myostatin protein or by transfecting cells with sense or antisense myostatin expression con structs These studies have demonstrated that myostatin is capable of blocking 19 Apr 2004 2125 AR AR2267CB20703tex AR2267CB20703sgm LaTeX2e20020118 P1 GCE AR EVIEWS 1N ADVANCElO l l46annurevcelbio20012103135836 MYOSTATIN 75 both proliferation Thomas et al 2000 Rios et al 2001 Taylor et al 2001 Joulia et al 2003 and differentiation Langley et al 2002 Rios et al 2002 Joulia et al 2003 of C2C12 myoblasts The effects of myostatin on cell proliferation appear to be mediated by its ability to increase levels of p21 decrease levels of Cdk2 and decrease phosphorylation of Rb Thomas et al 2000 Rios et al 2001 Myo statin appears to inhibit myogenic differentiation by downregulating expression of the myogenic regulators MyoD myogenin and Myf5 Langley et al 2002 Rios et al 2002 Joulia et al 2003 The effects of myostatin on myoblast pro liferation and differentiation do not appear to be restricted to C2C12 myoblasts as effects on cell proliferation have also been demonstrated in primary bovine fetal myoblasts Thomas et al 2000 and implantation of myostatincoated beads into developing chick limb buds has been shown to cause downregulation of Pax3 Myf5 and MyoD expression and a consequent decrease in the amount of limb muscle formed Amthor et al 2002b Taken together these studies suggest that myostatin normally regulates the number of muscle bers that are ultimately formed by regulating both proliferation and differentiation of myoblasts during development The ability of myostatin to regulate ber size suggests that a major target for myostatin signaling in adult animals is likely to be the satellite cell Satellite cells which are mononucleated cells located between the sarcolemma and the basement membrane of individual muscle bers function as a stem cell population in muscle for reviews see Campion 1984 Bischoff 1994 Schultz amp McCormick 1994 During muscle ber growth the normally quiescent satellite cells reenter the cell cycle proliferate and fuse with existing muscle bers The fact that loss of myostatin can cause increased ber sizes suggests that myostatin may normally act to suppress satellite cell proliferation andor differentiation This role for myostatin has been substantiated both by in vitro studies examining the effects of myostatin on satellite cells in culture and by direct analysis of satellite cells in myostatin mutant mice McCroskery et al 2003 Studies with isolated satellite cells have demonstrated that myostatin is capable of upregulating p21 expression down regulating Cdk2 expression and inhibiting cell proliferation Consistent with this activity of myostatin on satellite cells in vitro muscles of myostatin mutant mice have been shown to have an increased number of satellite cells per unit length and a higher proportion of activated satellite cells than muscles of wildtype mice Moreover satellite cells isolated from myostatin null mice appear to have a higher rate of proliferation in culture than satellite cells isolated from wildtype mice An appealing model is that the normal function of myostatin in adult muscle is to maintain satellite cells in a quiescent state and that in circumstances in which L 39 i lr iplirPI l thi 39 39 activity is inhibited thereby releasing satellite cells from growth arrest Although there is considerable evidence that myoblasts and satellite cells are targets for myostatin signaling in vivo it seems likely that other cells are also capa ble of responding to myostatin In particular overexpression of myostatin in adult mice has been shown to induce a dramatic systemic wasting syndrome cachexia 19 Apr 2004 2125 AR AR2267CB20703tex AR2267CB20703sgm LaTeX2e20020118 P1 GCE AR EVIEWS 1N ADVANCElO l l46annurevcelbio20012103135836 76 LEE characterized by extensive loss of muscle and adipose tissue mass Zimmers et al 2002 The rate and extent of muscle loss in these animals cannot be explained as an effect of myostatin solely on satellite cell function and raises the possibility that this effect may be mediated by myostatin signaling directly on myotubes consistent with this possibility is the observation that myostatin is capable of in hibiting protein synthesis in myotubes derived from differentiation of C2C 12 cells in culture Taylor et al 2001 In addition the rapid depletion of fat stores seen upon overexpression of myostatin in mice raises the possibility that myostatin may also have direct effects on adipocytes in this regard myostatin is capable of blocking adipogenic differentiation of both 3T3L1 and C3H 10T12 cells in culture Kim et al 2001 Zimmers et al 2002 Rebbapragada et al 2003 although effects of myostatin on fully differentiated adipocytes have not yet been reported Alternatively it is possible that the effects on muscle mass andor fat stores seen upon overexpression of myostatin may re ect the activity of some other mediator of cachexia whose production or activity is induced by myostatin 1f the effect of myostatin is indirect the direct mediator is unlikely to be TNFot or lL6 which are known to induce cachexia in mice Oliff et al 1987 Black et al 1991 as serum levels of r 39 d appear t d in 39 overexpressing mice Zimmers et al 2002 Although this depletion of fat stores was observed in mice in which the serum concentrations of myostatin were arti cially elevated it is clear that myostatin can in uence fat metabolism and adipocyte function even under normal conditions Mice lacking myostatin have a reduction in total body fat which is particularly pronounced in older animals Lin et al 2002 McPherron amp Lee 2002 The re duction in fat stores is contrary to what one might predict based on the effects of overexpressing myostatin in mice and on the effects of myostatin on adipogenesis in vitro One possible explanation to reconcile these ndings might be that the rapid depletion of fat stores seen upon overexpression of myostatin results from inappropriate direct signaling of myostatin on adipocytes whereas the reduction in fat accumulation in myostatin mutant mice might represent an indirect effect of the profound metabolic changes resulting from loss of myostatin signaling in muscle Clearly determining whether myostatin plays a direct or indirect role on adipo cyte development or function and whether myostatin can act directly on myotubes to regulate ber growth independently of its effect on satellite cells will be essential for understanding the full range of myostatin activities under both normal and pathologic conditions De nitive experiments to identify the cells that are direct targets for myostatin will almost certainly require conditional transgenic and gene knockout approaches to block myostatin signaling in a cell typespeci c manner in v1vo Clinical Applications The biological functions of myostatin have raised the possibility that targeting the myostatin pathway may be an effective strategy for increasing muscle growth 19 Apr 2004 2125 AR AR2267CB20703Jex AR2267CB20703sgm LaTeX2e20020118 P1 GCE AR EVIEWS lN ADVANCElO l l46annurevcelbio20012103135836 MYOSTATIN 77 for a variety of clinical applications Given that satellite cells may be targets for myostatin signaling in adult animals there has been considerable interest in the possibility that inhibition of myostatin may have bene cial effects in terms of y v v 1 L FIUJUULIII p degeneration such as in sarcopenia which is the progressive muscle loss that occurs in the elderly for review see Hepple 2003 or in patients with muscle degenerative diseases such as muscular dystrophy In this regard two studies Wagner et al 2002 Bogdanovich et al 2002 have investigated the effect of loss of myostatin activity in mdx mice which carry a mutation in the dysl39rophin gene Sicinski et al 1989 and have therefore been used as a mouse model for Duchenne muscular dystrophy Wagner et al 2002 analyzed the effect of the myostatin null mutation in mdx mice and Bogdanovich et al 2002 analyzed the effect of administering myostatin neutralizing monoclonal antibodies to mdx mice Perhaps not surprisingly both studies showed that loss of myostatin activity led to an increase in muscle mass and muscle strength in mdx mice as in wild type mice However the bene cial effects of loss of myostatin activity seemed to extend beyond simply enhancing muscle mass In particular although the muscles of these mice did exhibit some of the dystrophic changes typical of mdx mice loss of myostatin led to a signi cant histological improvement of the muscle including decreased brosis suggesting that inhibition of myostatin activity may actually enhance the regenerative capacity of muscle in the setting of repeated cycles of degeneration In this respect one concern is that blocking myostatin activity might rapidly deplete the satellite cell pool and thereby prematurely exhaust the ability of 39439 imlbyL quot 39 An encouraging nding however was that the increased muscle mass and decreased brosis were seen even in 9month old myostatinnull mdx mice suggesting that the bene cial effects of loss of myostatin could be maintained for at least this time period Wagner et al 2002 In addition to L 39 f 39 39 taroetino mvostatin may also have bene cial effects in disease states in which muscle loss is more acute One example is the muscle loss that occurs in cachexia which is a wasting syndrome often seen in patients with chronic diseases such as cancer AIDS and sepsis and which is a major contributor to both morbidity and mortality for review see Tisdale 1999 Although the effects of inhibiting myostatin activity in models of cachexia have not yet been reported it seems plausible that blocking this pathway might be an effective method for enhancing muscle growth or regeneration to offset the wasting that occurs in this syndrome Moreover as discussed above overexpression of myostatin in mice can induce a wasting syndrome that has many of the hallmarks of human cachexia including fat and muscle loss in the setting of normal caloric intake Zimmers et al 2002 This nding raises the intriguing possibility that either myostatin itself or some other mediator capable of activating this same signaling pathway may play a critical role in inducing cachexia in humans If so targeting this pathway may directly counteract the molecular signals responsible for inducing cachexia in various disease states In this respect 19 Apr 2004 2125 AR AR2267CB20703Jex AR2267CB20703sgm LaTeX2e20020118 P1 GCE AR EVIEWS lN ADVANCElO l l46annurevcelbio20012103135836 78 LEE circulating levels of myostatin have been reported to be elevated in HIVinfected patients with muscle wasting GonzalezCadavid et al 1998 although one caveat to the interpretation of these studies is the uncertainty regarding the speci city of the antibodies used to measure myostatin levels Finally targeting the myostatin pathway may have applications not only for muscle degenerative and wasting conditions but also for metabolic diseases such as obesity and type II diabetes As discussed above myostatin mutant mice fail to accumulate fat as a function of age Lin et al 2002 McPherron amp Lee 2002 This observation suggested the possibility that loss of myostatin might have bene cial effects in settings of abnormal fat accumulation Indeed introduction of the myostatin null mutation into two different obese strains agom i lethal yellow and Db0b resulted in a signi cant reduction in fat accumulation and suppression of the development of insulin resistance NcPherron amp Lee 2002 Although the precise mechanisms by which myostatin regulates fat and glucose metabolism in vivo are not completely understood these ndings raise the possibility that inhibition of the myostatin pathway may be a new strategy for the prevention or treatment of obesity or type II diabetes Clearly much more work will be required to determine whether targeting the myostatin pathway will have bene cial effects in settings of human diseases A major unanswered question is whether myostatin plays a role in regulating muscle mass in humans as it does in mice and cattle although the high degree of sequence conservation and expression of myostatin in human muscle are certainly consistent with a similar role for myostatin in humans lf targeting this pathway does turn out to be an effective strategy for treating human diseases a number of consider ations make myostatin a particularly attractive target for drug development First the effects of loss of myostatin are highly speci c as signi cant effects on tissues other than muscle and adipose tissue have not yet been reported Second myo statin normally functions extracellularly and is therefore accessible to a variety of potential pharmacologic agents including proteins capable of binding myostatin similarly the mechanisms involved in regulating myostatin latency also operate extracellularly making them readily accessible as well Third the effect of myo statin is dose dependent in that partial inhibition of myostatin activity results in a partial increase in muscle growth hence it may not be necessary to completely block myostatin signaling in order to produce a therapeutically bene cial effect CONCLUSIONS Although many details regarding the mechanism of action of myostatin and the mechanisms by which myostatin activity is regulated in vivo remain to be eluci dated it is now rmly established that myostatin is synthesized by skeletal muscle circulates in the blood and acts in a concentrationdependent manner as a nega tive regulator of muscle growth These properties of myostatin are precisely those hypothesized by Bullough 1962 1965 for molecules that act to regulate tissue 19 Apr 2004 2125 AR AR2267CB20703tex AR2267CB20703sgm LaTeX2e20020118 P1 GCE AR EVIEWS 1N ADVANCElO l l46annurevcelbio20012103135836 MYOSTATIN 79 size and to be consistent withthe terminology that he proposed it seems clear that myostatin should be considered to be a muscle chalone Of all the tissues that could potentially utilize this type of mechanism to regulate tissue size it is perhaps a bit unexpected that the one tissue for which this type of regulatory mechanism has been established is skeletal muscle After all growth of individual muscles is known to be controlled locally in response to a variety of different environmental stimuli and stresses including exercise and injury How then does one reconcile this type of local control with the fact that a key regulator like myostatin circulates systemically in the blood Almost certainly a critical aspect of the biology of myostatin is that myostatin circulates in the blood in a complex with inhibitory proteins and is therefore biologically inactive It seems reasonable to suppose that levels of myostatin activity are regulated primarily at the target site by local control of the mechanisms involved in activating myostatin latenc However even if local signals do dictate the nal level of myostatin signal ing in muscle it is possible that circulating levels of myostatin are regulated to limit the overall capacity for muscle growth A speculative model is that circulat ing levels of myostatin change under different environmental conditions such as temperature or food availability or under different physiological states such as physical activity illness pregnancy or age and that these changing levels then alter the metabolic homeostatic balance between fat storage and muscle growth Under certain conditions it may be advantageous to shift this balance toward fat storage and under different conditions it may be advantageous to shift this bal ance toward muscle growth Numerous studies have examined the regulation of myostatin expression in response to a variety of stimuli and under different physio logical conditions and upregulation or downregulation of myostatin expression was detected in many of these studies GonzalezCadavid et al 1998 Ji et al 1998 Carlson et al 1999 Sharma et al 1999 Lalani et al 2000 Mendler et al 2000 Sakuma et al 2000 Wehling et al 2000 Kawada et al 2001 Lang et al 2001 Ma et al 2001 2003 Marcell et al 2001 Schulte amp Yarasheski 2001 Brill et al 2002 Kocamis et al 2002 Tseng et al 2002 Welle et al 2002 Yarasheski et al 2002 Armand et al 2003 Chauvigne et al 2003 Jeanplong et al 2003 Liu et al 2003 Pampusch et al 2003 Peters et al 2003 Rodgers et al 2003 Roth et al 2003 Vianello et al 2003 White et al 2003 however additional ex periments will be required to determine whether the magnitude of these observed changes is large enough to cause signi cant physiological effects Although we still have much to understand regarding the biology of myostatin it is clear that it myostatin is a major regulator of the metabolic state of the animal and it is the pro found metabolic consequences resulting from altered myostatin activity that likely explain the extraordinary degree to which myostatin has been conserved through evolution It remains to be determined whether myostatin is unique as a negative regulator of muscle growth or whether the activity of myostatin is at least partially redun dant with those of other signaling molecules such as the highly related protein 19 Apr2004 2125 AR AR226VCB20703tex AR2267CB20703sgm LaTeX2e20020118 P1 AR GCE EVIEWS IN ADVANCE 10 1 146annurevcellbio200 12 103 135836 80 LEE GDFll It will also be important to determine whether this general type of regu latory mechanism may be utilized by tissues other than muscle to regulate tissue size Because activation of the TGF signaling pathway results in growth inhi bition in a variety of cell types and because the TGF superfamily contains an enormously large group of ligands with a wide range of expression patterns this family of signaling molecules may be a reasonable starting point in the search for other chalonelike modulators of tissue size ACKNOWLEDGMENTS I thank Christine Moss for assistance with preparation of this manuscript I apol ogize to those colleagues whose work I did not cite as a result of either space limitations or oversight Work in my tional Institutes of Health grants R01 laboratory on myostatin is supported by Na HD35887 and R01CA88866Myostatin was licensed by the Johns Hopkins University to MetaMorphix Inc and sublicensed to Wyeth I am entitled to a share of sales royalty received by the University from sales of this factor The University a subject to certain restrictions under nd I also own MetaMorphix stock which is University policy I am a paid consultant to MetaMorphix The terms of these agreements are being managed by the University in accordance with its con ict of interest policies The AnnualReview of Cell and Developmental Biology is online at httpcellbioannualreviews0rg LITERATURE CITED Alciyoshi S Inoue H Hanai Ji Kusanagi K Nemoto N et all 1999 cSki acts as a transcriptional corepressor in transforming growth factor signaling through interac tion with smadsi J BZOl Chem 2741352697 77 Amthor H Christ B RashidDoubell F Kemp CF Lang E Patel Ki 2002a Follistatin regu lates bone morphogenetic protein7 EMF 7 activity to stimulate embryonic muscle growth Dev Biol 24339115727 pression and regulation of follistatin and a fol l i Stall l ike gene during avian somite com partrnentalization and myogenesisiDev BZOl 1782343762 Amthor H Huang R McKinnell I Christ B Kambadur R et al 2002b The regulation and action of myostatin as a negative reg ulator of muscle development during avian embryogenesisi Dev BZOl 2512241757 Armand AS 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