The Proteoglycan Perlecan Regulates Long Bone Growth Through

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Graduate Theses and Dissertations Graduate School

2007 The proteoglycan perlecan regulates long bone growth through interactions with developmental proteins in the growth plate Simone Marsha-Lee Smith University of South Florida

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Scholar Commons Citation Smith, Simone Marsha-Lee, "The proteoglycan perlecan regulates long bone growth through interactions with developmental proteins in the growth plate" (2007). Graduate Theses and Dissertations. http://scholarcommons.usf.edu/etd/2370

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G r o w t h P l a t e

b y

S i m o n e M a r s h a - L e e S m i t h

A d i s s e r t a t i o n s u b m i t t e d i n p a r t i a l f u l f i l l m e n t of the requir eme nts for th e degr ee of Doctor of Phi los ophy Depar tment of Mol ecul ar M edi cine Coll ege of Medi cin e Univer sit y of South Flori da

Maj or Profes sor: Joh n R. Ha s sell, Ph. D. Paul Gott s chal l, Ph. D. Willia m R. Gow er, Jr. , Ph. D. David Morg an, Ph. D.

Date of A pprov al July 6, 20 07

Keyw ords : carti lag e, endo cho ndral, fgf , fgfr, hspg2

Dedic ation

This di ss ertat ion i s for my family i n Ja maic a, here in th e U. S. and ev eryw here e ls e. My p arent s, si st e rs, brother, ni ece and al l the family an d friend s wh o ins pire a nd s up port me, I dedic at e this t o you. Rem emb er- “The biggest adventure you can ever take is to live the life of your dreams. Think like kings and queens, not afraid to fail; failure is another stepping-stone to greatness”. Oprah!

Ackno wledg ement s

I could ne ver ha ve c omple ted th is di s s ertatio n with out the supp ort of my famil y. My par ent s, Aub rey and Ja ckie, I am gra teful for the ac cep tan ce yo u hav e sh own e ve n when y ou prob ably di dn’t know ho w or whe n gradu ate sc hool for me would e nd. My si ster s, San and Sh en a nd my broth er Ki sh, yo u are (almo st) al l grown u p and I can not w ait to s ee w hat yo ur fut ures w ill bring. I am s o thankfu l for (and prou d of) the thre e o f you. My be autifu l and brillian t nie ce, Sera yah, a mill ion th an ks for the u nexp ec ted bu t invalu abl e life le s son s yo u hav e taug ht all of us. My me ntor, Dr. Ha ss ell, tha nk you for your exp ert guid an ce over the y ear s. Bra d Kan e, it ha s reall y been fu n in the l ab; tha nk you for all your h elp. To former H as se ll lab mem ber s Bridg ett e, Kurt, Leigh an d Pras ant hi, I appre ciat e every s ec ond th at ea ch of you sp ent to entert ain, gui de, hel p, an d (esp eci ally Pra s anthi) instru ct me. To Debbi e, Hele n, Kath y and Su s an, th ank you for b eing so comp etent and c aring. To the pr ofe ss ors (es pe ciall y Drs. B lan ck, Gott sch all, Gow er, Kell er, Morg an an d Solomon son), for your h elp over the y ear s, than k you al l.

“Science has great beauty. A scientist in her laboratory is not only a technician: she is also a child placed before natural phenomena which impress her like a fairy tale”. Marie Curie. French (Polish-born) chemist & physicist (1867 - 1934)

Note to Re ade r

The origin al of thi s doc ument cont ain s color that i s ne ce s sary for under sta nding t he da ta. The origi nal di ss ertat ion i s on file w ith the USF library in Tam pa, Florid a.

Table of C ontent s

List of Figure s ...... iii

Abstr act ...... vi

CHAPTE R ONE : INTRO DU CTION The Growth Plat e and Lon g Bon e Growth ...... 1 How Longitudi nal Bone Growth O ccur s ...... 12 Perlecan ...... 17 Fibroblast Growth F act or (FGF)-2 ...... 27 FGF-18 ...... 29 Histone H3 ...... 32 Outline ...... 34 Referen ce s For Introdu ctio n ...... 36

CHAPTE R TWO : PAPERS Paper I ...... 48 Paper II...... 83 Paper III ...... 110

CHAPTE R THREE : CON CLUSI ONS Conclu sion s ...... 131

Refer enc e s ...... 144

Appe ndix : Fo cus o n Perle ca n Focus on Perl ec an ...... 162

About th e Aut hor ...... End Page

List of Figu re s

Figure 1. Location and org aniz atio n of the growt h plat e ...... 2 Figure 2. Structure of p erlec an, s ho wing it s 5 dom ain s and the atta ched GAG s ...... 18 Figure 3. Bios ynth es is of Perl ec an ...... 19 Figure 4. Morphol ogy of wil d-typ e (a, c, e) versu s h spg 2 null (b, d, f) mice ...... 26 1 2 5 Figure 5. Bindin g of [ I]-FGF-2 to perle can in t he cationic filtrat ion (C AF) a ss ay ...... 69 Figure 6. Effect of chondr oitin as e or hepariti na se pretreat ment of p erle can o n sp ecifi c b inding 1 2 5 of [ I]-FGF-2 in the CAF as say ...... 70 Figure 7. Effect of chondr oitin sulfa te or hep aran sulfate on s pe cific FGF- 2 bindin g in the CAF ass ay ...... 71 1 2 5 Figure 8. Bindi ng of [ I]-FGF-2 to perle ca n in the immunopr eci pitat ion (IP) as s ay ...... 72 Figure 9. Effects of ch ondroi tina s e a nd hep aritin as e 1 2 5 pretreat ment of p erle can o n [ I]-FGF-2 binding u sin g the IP a ss ay ...... 73 Figure 10. Bind ing of FGF2 t o sol ubl e FGFR-1 c and FGFR-3c ...... 74 Figure 11. Hep arin an d perl eca n aug menta tion of [ 3 H]- thymidin e inc orporat ion int o FGF-2 tr eate d BaF3 c ell s ...... 75 iii Figure 12. Hypot heti cal m odel of FG F-2 binding t o perle ca n and delivery to FGF r ece pt or ...... 76 1 2 5 Figure 13. Satura tion bi nding of I- FGF-18 to grow th plate perlec an in th e ca tio nic filtrat ion (C AF) assay ...... 99 Figure 14. Effect of cho ndroit ina se o r hepariti na se 1 2 5 pretreat ment of gro wth pl ate p erle can on I- FGF-18 binding us ing th e CAF a s say ...... 100 Figure 15. Effect s of chon droitin a se and he pariti na se 1 2 5 pretreat ment of p erle can o n I-FGF-18 bindin g using the immun opre cipit a tion (IP) as sa y ...... 101 Figure 16. Effect of growt h plat e perl eca n core pro tein denaturatio n and re duct ion /alkyl atio n on 1 2 5 I-FGF-18 binding ...... 102 1 2 5 Figure 17. Bind ing of I-FGF-18 to nativ e full-le ngth perlec an an d to rec ombin ant p erle can domain s I-III in the CAF a ss ay ...... 103 1 2 5 Figure 18. Bind ing of I-FGF-18 to different p erle can domains in th e immuno pre cipit ation (IP) a ss ay . .. . 104 Figure 19. Affinity p urific ation of p e rleca n’s bi nding partners ...... 124 Figure 20. Cat ioni c Filtrati on (C AF) As say s show t hat perlec an bin ds to 1 2 5 I-hi ston e H3 ...... 125 Figure 21. Effect of cho ndroit ina se a nd hep aritin as e pre-treatm ent on t he bin ding of p erle c an to 1 2 5 I- histo ne H3 ...... 126 Figure 22. Immunol oca liz ation of Hi s tone H3 and Perlecan in the d ev elopi ng growth pla te ...... 127 Figure 23. Differring affiniti es of FG F-18 and FGF-2 for Growth Plate Perl eca n ...... 138 iv Figure 24. Perle can core prot ein: D et ermine d domai n structure an d GAG atta ch ment ...... 168

The Proteog lyc an Perl ec an R egul ate s Long Bo ne Gro wth throu gh Interac tion s wit h Re gula tory Prote in s in the De velo ping Gr owth Plate

Simone Mar sha-L ee Smith

ABS TRA CT

Perlec an i s the m ajor he para n sulf ate p roteogl yca n (HSPG) in growth pl ate c artil age and i s criti cal f or growth pl ate c hondro cyt e proliferat ion a nd prop er sk elet al de vel opment. It s core pr otei n and atta che d chon droitin sulf ate (C S) and hepar an s ulfat e (HS) ch ain s media te int erac tion s wit h many di ver se protein s. Fibrobl as t growth factor (FGF)-2 a nd FGF-18 are oth er regul ators of chon drocy te proliferat ion in th e growt h plat e. Addi tionall y, FGF-18 co ntrol s the hypertro phy a nd cart ilag e va sc ulari zati on nec e ss ary for endo chon dral o ss ific ation. The re se arc h pres ent ed in thi s dis sert ation aime d to ide ntify kno wn a nd nove l perle ca n-bindi ng protein s th at are e ndog eno us to t he gro wth plat e an d to ch aract eriz e their int eract ion s with p erle can. FGF-2 (know n to bind HS PG s) bound t o perle can in b oth a catio nic filtr ation ( CAF) a s say a nd an immunopr eci pitat ion (IP) as sa y primaril y via th e HS ch ain s on p erlec an. Wh en dig es ted wi th chondr oitin as e AB C to rem ove it s CS c hain s, perle ca n augm ent ed bindin g of FGF-2 to the F GFR-1 and F GFR-3 re cept ors and incre as ed FGF-2 - stimul ate d prolifer at ion in B aF3 ce lls expre s sing

vi the se FGF re cep tors. Thu s, growth p lat e perle can b ind s to FGF-2 b y its HS c hain s but can o nly de liver FGF -2 to FGF rece ptor s whe n its CS ch ain s are rem ove d. FGF-18 (kno wn to bin d to hep arin a nd to hepar an sulfat e from som e sour ce s) bou nd to grow th pla te p erlec an. This b indin g wa s unch ang ed by ch ondroi tina s e or hep ari tina se dig e stion of p erle can, indic ating t hat p erlec an G AG s are no t involv ed in FGF-1 8 bindi ng. FGF-18 bou nd equ ally t o recom bin ant domain s I-III of perle can (Alt1) and to fu ll-len gth perl ec an purif ied from the gro wth pl ate. Additi onall y, FGF-18 bo und eq uall y to recombi nant d omai n III of perle can, to Al t1 and t o Alt2 (a d omai n I-III variant with n o hep aran sulfa te). Therefor e, bindin g sit e s for FGF-18 are pr es ent in d omain III of perleca n. Affinity c hromat ograp hy i solat ed hi sto ne H3 a s a p erlec an- bindin g protei n from the c hondro cyt e matrix. CAF as s ays confirm ed the int eract ion a s s pe cific, de pen dent primarily o n HS ch ain s of perle can, alt houg h CS c hain s an d the p erlec an cor e wer e al so involv ed. Immuno his toch emi stry d ete c ted perl ec an an d hist one H3 colo cali zed in gr owth pl ate cartil ag e. Thes e res ult s can h elp u s better u nder sta nd the gr owth fa ctor-in depe nde nt contr ol that perle can e xert s on e ndoc hondr al o ssif i catio n and, th erefore, lo ng bone gro wth.

vii

CHAPT ER ON E

IN TROD UC TI ON

The Gr owth Plate and Long Bone G r owth The growth pl ate i s th e princi pal re gul ator of longi tudin al bone gro wth in hu man s an d anim al s. Longitu dinal gr owth i s controll ed by m ultipl e env ironm enta l, gene tic, hormo nal, an d nutrition al fa ctor s. Togeth er, thes e fac tors de termin e ad ult hei ght by influe ncin g the pro liferat ion a nd di fferenti ation of c hondr ocyt e s in the grow th pla te. The regi on of cart ilage k nown as th e grow th plate i s pre se nt only i n bon es th at for m through a cartil age precur sor, that i s, by en doc hondr al o ss ificati on. In the se endo chon dral bo ne s (su ch a s the m andi ble, ribs, limb s a nd digit s) a growth pl ate i s si tuat ed b etwe en th e ep iphy sea l and m etap hy sea l region s of bon e at th e dis tal e nd s of th e long bo ne s (Figure 1 *).

The Gr owth Plate 1a. Origin The formatio n of chon droc yte s (cho ndr ogen esi s) i s a high ly regula ted pro ce s s. The cho ndropro geni tor cell s are m es en chym al stem cell s (MS C s). The MS Cs aris e fro m different lo cat ion s depe nding o n their t arget s in e mbryog e nes is. For ex ampl e, cell s th at will be come chon drocy te s in vert ebra e and ribs ori gin ate from th e paraxi al me sod erm wh ere as c ell s de sti ned for limb c artil age

1 originat e from the l ater al me sod erm (d e Crombru ggh e, Lefebvr e et al. 2000). In the em bryo, en doch ondr al oss ifica tion in itiat e s with MSC cond en sati on (that i s, the MSC s f orm clu ster s and adh ere via adhe sio n mole cul es) (H all an d Miy ak e 2000) an d progre s se s to th e sub se que nt differe ntiat ion of th e MS Cs into chon drog enic cell s. Even after d eve lopm ent en ds, MSC s ar e know n to per si st a s st em cell s in th e bone m arrow of a dult ma m mals, w here th ey ret ain th e cap acit y to be come chon drocyt e s (Ma g ne, Juli en et al. 2005).

Figure 1: Loc ation and org aniz atio n o f the growth pl at e. *From van der Eerden, B. C. J. et al. Endo cr Re v 2003;2 4:782- 801

As in a dipog en esi s, myo gen esi s or o ste ogen esi s, MS C different iatio n into c hondr ocyt e s is c o ntrolled b y DN A tran scrip tion factor s. In chon droge ne si s, this fa ctor is Sox9 (O ls en, Re gina to et al. 2000). MS Cs, in re sp on se to TGF-ß and fibron ecti n, start forming pre chon drocy tic c onde ns atio n s at differ ent site s in th e embryo. Thi s occ urs a t aroun d mid-g es tation (~E1 0.5) in mice

2 (Lefebvre, Li et al. 19 98). By E12. 5, c artilag e is form ing a ctiv ely in all de stin ed c artila gino us ( suc h as ear and no se c artil age) a nd endo chon dral (s uch as rib a nd limb b o ne) stru cture s. In intra me mb rano us o s sific ation (w hic h g ive s rise t o bon es suc h as t he flat bon es of th e sk ull), the c ond ens ed cell s differ enti ate int o bon e- forming o steo bla st s, whic h then se cret e a bony m atrix ric h in type 1 colla gen. Ho wev er, in the mor e comm o n endo chon dr al os sifi cati on, the ce lls i n the mid dle of th e con den sa tion clu st er bec ome chondr ocyt e s and c ell s on th e border f orm a peric hondri um. Scient ist s fir st reali ze d the imp ortan ce of Sox9 in chon drog ene si s on dis cov ering So x9 mut atio ns a s th e e tiology for c ampo meli c dysp la sia ( CD). CD i s a rare, sev ere, l ethal d warfi sm in hum an s (Foster, Do mingu ez-St egli ch et al. 199 4; Wagn er, Wirth et al. 1994). CD p atie nt s hav e abn ormalit ie s in es se ntia lly all cartil age- derive d bon es in clud ing th e cran iofa ci al bone s, vert ebra l colu mn, ribs an d limb s. The bow ed, ang ulat ed bone s an d other ske let al defec ts of CD pat ient s re sult from d efe ctive chon drocy te different iatio n withi n the MSC cond en sati on s and th e re sulti ng defici ent pro ducti on of ch ondro cyte s. The Sox9 g en e is ubi quito us i n chon dr oproge nitor c ell s and in the cho ndro cyte s th ey be com e (de Cro mbruggh e, Lefebvr e et al. 2000). Sox9 c onta ins a DNA b indin g d omain a nd a strong trans cripti on a ctiva tion do main, m akin g it a pote nt tran scri ption factor th at bind s to th e gen e s for chon drocy e-sp ecifi c mark ers su ch as c olla gen Typ e II and ag grec an. If Sox9 is mu tat ed or ab se nt in mous e mod els t he se mar ker prot ein s ar e not expr e ss ed in th e MS C and a blo ck o ccur s at th e poin t wher e cond en sati on woul d norma lly occur (d e Crombr uggh e, Lefebvr e et al . 2000), making th e c ells unabl e to differ enti ate int o cho ndrog e nic pre curs ors, ev en th ough their migra tion to th e wo uld-be cond en sati on si te i s unaff ect ed (Bi,

3 Deng e t al. 1999). The p erich ondriu m is the o nly fina l loc ation of suc h Sox9 nu ll cel ls; t he MS C s thu s m igrate to t he co nde ns atio n cent er fine wit hout So x9 but do n ot dif ferenti ate int o cho ndroc yte s withou t Sox9 drivi ng it s cho ndroc yte- s pecifi c gen eti c program.

1b. Growth Pl ate Stru cture The growth pl ate cont ain s only o ne typ e of cell, th e chondr ocyt e. How ever, th e cho ndroc yt es ar e of three d ifferen t su b- type s com prisi ng th e three d ist inct h orizont al lay ers of th e growt h plate cartil age, th e re sting (w here th e stem cell s ar e), proliferat ive and pre hypertr ophi c/hy pertrop hic z one s (Figure 1). The gro wth pl ate cartil age e xi sts o nly duri ng the gr owth period of mo st ani mal s, for exam ple, up to pu berty i n huma ns. Aft er the grow th perio d, the cartil age i s com plet ely re sorb ed a nd re plac ed by bo ne. The three t ype s of cho ndro cyte s in th e growth pl ate ar e bioch emic ally, fun ctio nally and morp h ologic ally d ist inct. The mo st epiph ys eal of th e zon e s is th e re sting z one. Her e, chondr ocyt e s are smal le st, rarely mi totic, a nd are arrang ed irregul arly in th eir s par se cartil age m atrix. The re stin g zon e cel l s are rel ativ ely qu ie sc ent but they ar e cruci al for prop er arrang em en t of the other u nderl ying column s of c ell s and th us, for promo ti on of unidire ctio nal longitu dina l bon e growth. The re sti ng zone i s tho ught to cont ain stem-l ike c ell s with a unip oten t abilit y to gener ate n ew c ell s of the proliferat ive zone (H unzi ker 19 94). On som e yet u nkno wn s timulu s, th e mo re mature re sti ng c ell s divide t o produ ce tw o cell s; o ne ret ain s the stem-l ike re sti ng ce ll pheno type and th e oth er bec ome s proli ferativ e. Upon di vis ion of a cell in th e prolif erativ e zo ne (a c ell th at is no w five tim e s bigg er than a re sti ng ce ll (Noon an, Hun zik er et al. 1998)) th e ne wly forme d clon e line s up p arall el to the l ong a xis of the bone. Thi s re sult s in

4 expa nsi on of col umn s of cel ls ori ent ed along th e leng th of the b one, leadi ng to bo ne el onga tion. Thi s alig n ment of the pr olifer ativ e cell s into col umn s para llel to t he el onga tion directio n of the bo ne i s direct ed by a m orphog en synth e siz ed b y resti ng ce lls (A bad, Mey ers et al. 2002). In the pr olifer ating zone, the ce lls n ot only align i nto column s, but th ey al so fl atte n, divid e rapidly and synth e siz e larg e amount s of ex trac ellu lar matri x or EC M (up to 46% mor e than resti ng ce lls) w hich i n turn mai ntai ns t he stru ctur e of the gro wth plate. The stim ulu s for prolifer ating chon dro cyte s to progr e ss through th e final, m ature ph a se of th e hypertro phic zon e is tho ught to be exp os ure to lo cal gro wth fa ctor s in the grow th pla te. The Indian h edg ehog/P arat hyroid h ormon e- relate d pep tide (Ihh /PTHrP) pathw ay c au se s prolif eratin g cho ndroc yte s to sto p divid ing a nd st art different iatin g to ent er the n ext ph a se: hypertrop hy (Mi nina, Kres ch el et al. 20 02). With thi s ph as e als o com es a m arke d incre a se in cell siz e (a ce ll that i s up to t en tim es th e si ze of a re sti ng ce ll) (Noona n, Hunz iker et al. 1998). Thi s transi tion e vent uall y res ult s in larger, round, hy pertro phic chon drocyt es th at s ecr ete v ast amoun ts of ECM (up to 60% m ore tha n a re stin g cell) (No onan, H unzi ker et al. 1998), intra cell ular c alc ium an d ve sicl e s cont aini ng ca lciu m– phos pha te s, hydrox yap atite and ma trix metallo prote ina se s wh ich a ll cau se mi neral iza tion of th e carti lag e ECM in t his h ypertro phi c zon e (Nils son, Marin o et al. 200 5). Min erali zatio n ca us es bl ood v es se ls to enter from th e bon e adj ac ent to th e growth pl ate. The v es s els bring in o steo bla st s and o st eoc la st s: th e ost eoc la st s degr ade th e struc ture s tha t previ ous ly ke pt the hy p ertrophi c cho ndroc yte s arrang ed in co lumn s whil e the o st eobl a st s remod el the sc affold creat ed by th e ap opto se d, mineral ize d hypertro phic chon drocy te s into ne w meta phy se al bon e. With th is effecti ve d eleti on of the

5 termina lly differ enti ated h ypertr ophi c cell s an d the cr eati on of bone, the gr owth pl at e si ze i s maint ain ed whe n matur e prolif erativ e cell s st art to be com e hyp ertrophi c, mat ure res ting c ell s be come proliferat ive, an d more MSC s co nde ns e and diff erenti ate i nto ne w resti ng ce lls. Wi th thi s co ntinu al repl e nishm ent d uring per iod s of growth, the gr owth pl ate cartil ag e cau s es lo ngitu dinal gr owth of endo chon dral bo ne s.

1c. ECM Prot ein s From the re sting t o the hy pertrop hic z one, cho ndroc yte s are embe dded i n a matrix k now n as t he EC M, whic h is int egra l to bon e growth not o nly by it s she er ac cumul at ion but al so b y the v ariety of molec ule s tha t it cont ain s. Collag en s: The prolifer atin g cell s e xp res s primari ly coll ag en type II (Col2) w hile t he pre hyper troph ic cell s e xpre s s Col 9 and th e hypertro phic cell s ex pre ss Col1 0. Col2 and Col 9 are un ique t o cartil age (W ata nab e, Yam ada e t al. 19 98). Muta ting a ny of the se colla gen s c au se s wel l-ch aract eriz ed hu man cart ilag e matri x disord er s that a ll res ult in dw arfi sm. For examp le, muta tion s in human Coll age n II cau se skel eta l dis orders suc h a s chondr ody spl asi a s, epiph ys eal d ys pla si a and Sc hmid-ty pe metap hy sea l cho ndrody spl a sia (Spra ng er, Winterp ach t et al. 19 94; Mura gaki, Marim an et al. 19 96; W alli s , Rash et al. 199 6). Mut ation s in mous e mod el s hav e al so s hown t he i mportan ce of c olla gen to lo ng bone gro wth (Sta cey, Bat eman et al. 1 988; Gar ofalo, Vu orio et a l. 1991; Met sar anta, G arofal o et al. 199 2 ). Proteogly can s: C artila ge al so c onta in s vario us pro teo glyc an s suc h a s the c ell- surfa ce s ynd eca n and glypic an, the sma ll bigl yca n, decori n and lu mic an, and th e larg er ag greca n and p erle can (K nud son and Kn uds on 200 1). A proteo glyc an (P G) is com po sed of a core

6 protein w ith gly co sami nogl yca n (GAG) chain s co val ently atta che d (Has cal l, Cal abro et al. 1994). The dif ferent PG s ha ve var iou s typ es of GAG s att ach ed (for ex ample chon dr oitin, hep aran and ker ata n) and are all s ulfat ed (hen ce chon droitin sulfat e or CS, hep aran sulfa te or HS et c). The major c hondr oi tin sulf ate pro teog lyc an (CSPG) in c artila ge i s aggr ec an. The major he para n sulf ate proteog lyc an (HSPG) in cartil age i s p e rleca n, whic h is th e su bje ct of my dis sert atio n res ear ch and whic h I will dis cu ss in d eta il lat er. The role s of the PG s are t o bind c artil age prot ein s wit h their sulfa ted G AG grou ps and wit h their c o re protein s, ther eby cr os s- linking t he EC M. The su lfatio n of the s e PGs i s cru cial, th en, for cartil age in tegrit y, as i s s hown i n the many skel etal d ys pla si as th at resul t in dwarfi sm if di astr ophi c dy spl asi a su lfate tr an sport er (DTDST), a key PG s ulfati on en zym e in the growt h plat e, is mut ate d (Ros si a nd Sup erti-Furg a 2001). Al so, the di sorder h ered itary multipl e exo sto s es (H ME) occ urs from the mis- expre s sion of t he EXT1 or 2 gene s whi ch en cod e gly co sy ltran sfera se s th at s ynth esi ze hepar an s ulfat e GA Gs (Simm on s, Mu sy et al. 1999). H ME patie nt s pres ent wit h limb d eformiti es, le g leng th dis crep anci e s and ot her ske let al defe ct s all re sul ting from th e l os s of HS on th e chondr ocyt e s and th us lo s s of the se G AGs as c orec ept ors for grow th factor s, res ulting i n aberr ant prolif erat ion of growth p lat e chondr ocyt e s (for inst anc e, wher e HS: FGF bindin g is lo st) or of different iatio n of prolifer atin g to hyp e rtrophic c hondr ocyt e s (for inst anc e, wher e HS:Ihh bi ndin g is lo st ). The spec ific cor e prot ein s can al so re gul ate c artila ge-m aint ainin g proce ss e s su ch a s col lag en fibril formatio n (for decori n) and a nch oring of the c hondr ocyt e to its cy tos kel eton (for synd ec an) (Ba lloc k and O' Ke efe 200 3). Perlec an i s al so e s senti al for c artila ge integrit y and gro wth fa ctor sign alin g, as wi ll be di sc us se d lat er.

7 Aggre can a ls o play s a m ajor role in ca rtilage. Wi th it s ~230 kDa cor e prot ein an d up to 130 GAG c hain s (CS an d ker atan sulf ate or KS) aggre can can re ach a total m ole cular ma s s of ~22 00 kD a in som e tis su es (H as cal l, Cal abro et al. 1 994). Cartil ag e matrix defici enc y (Cmd) in mi ce i s a let hal c h ondrody spl a sia th at re sult s when th e ag grec an ge ne i s muta ted. Th e cmd mut ation i n the globul ar doma in I of aggre can i s le thal when hom ozy gou s, cau sin g early po stn ata l de ath from re spirat ory failure (R itte nhou se, D unn et al. 1978; W ata nab e, Kimat a et a l. 1994 ). These mic e, with 4 1% of the norm al aggr ec an mR NA, hav e s hort snout s, cl eft pal ate, s kel etal dwarfi sm an d mis- align ed s pin es du e t o the ab se nc e of aggre ca n in their car tilag e. The ch ondro cyte s in th e cmd nul l growth p lat es ar e irregularl y pa cke d toget her wit h rough collag en fibril s arra nge d around th em. This d isor gani ze s th e cel ls in co mpari son t o the orderly c olumn ar arran gem ent of ch on drocyt es in wild typ e. It is not surpri sing, th en, that t he se mi ce h ave r educ ed gro wth of the ir long bone s. Anoth er mutat ion in th e aggr ec an ge ne in chick en s re sult s in the cho ndrod ys pla sia k now n as n anom e lia (Li, Schwart z et al. 1993). The na nomel ic ch ick sho ws sim ilar carti lag e and b one defec ts as th e cmd mo us e, ie. dy smorp hic cart ilag e, dwarfi sm a nd letha lity. The mut ation i s in th e exo n c oding for the CS att ach ment region in aggre can, re sul ting in a tru nc ated pro tein th at c anno t acc ept C S cha ins. W ithou t thi s CS dom ain, aggr eca n ca nnot prom ote chondr ocyt e EC M inte grity an d the c ar tilag e prec ursor a nd thu s th e sub se que nt bon e is di srup ted. Growth Fact or s: The EC M is a re s erv oir of growth fa ctor s that are eith er mad e in the gro wth pl at e or trans porte d ther e, bound by other EC M prot ein s an d rele as ed to affect c hondr ocyt e ac tivit y (van der Eerd en, Karp erie n et al. 200 3 ). Insulin-lik e growt h fact or

8 (IGF)-1 exert s s ubs tant ial c ontrol ov er long bon e growth. IGF defici enc y ca us es f etal gro wth ret ardat ion and p ost nat al growt h failure in h uman s (C ama cho- Hubn er, Wood s et a l. 1999) an d mice (Moh an, Ric hman et al. 200 3), sin ce I GF-1 stim ulat e s both expa nsi on of prolif eratin g cho ndroc yte s and chon drocy te hypertro phy. The TGF-ß family of bo ne morp hog ene tic prote in s (BM Ps) ar e als o cruci al in bo ne gro wth, with rol es in skel eta l apop to sis, chondr ocyt e differ enti ation, forma tion of hypertrop hic c hondro cyt es and oth er a spe cts of sk eleto gen e sis ( M acia s, Ga nan et al. 1997). The BMP s an d their re ce ptor s are ex pre ss e d local ly in the gr owth pl ate and are n ec es s ary for proper c hondr og ene si s (Zou, Wie ser et al. 1997; Yi, D alui ski e t al. 2000). Ihh and PTHrP are o ther EC M mol ecu l es ma de lo call y in the growth pl ate. Ihh i s from the h edg ehog family of morpho gen s wit h cititi cal rol es in embryo nic form ation and de velo pmen t. Prehyp ertrophi c cho ndro cyte s e xpre ss Ihh, where it re gula te s the rate of cho ndro cyte d ifferen tiati on fro m proliferat ive to hypertro phic zon es (Vor tkam p, Lee et al. 1996). Blo ck ed hypertro phy re sult s if thi s Ihh i s mutat ed. St. Jac que s et al (St- Ja cqu es, Ha mmer sch midt et al. 1999) s howe d that Ihh- null mic e not only s how ed thi s redu ce d differe ntiati on of prolifer ative i nto hypertro phic cell s but w ere a ls o dwarf s with dr ast ic red ucti on s in chondr ocyt e prolif eratio n and o st eobl a st format ion. Ihh is t heref ore a key gro wth fa ctor for the c oupli ng of chondro gen es is a nd ost eoge ne si s (Karp, Sch ipa ni et al. 20 0 0). Ihh is an up stre am posit ive re gula tor of PTHrP and a ct s t hrough PTHrP to c ontrol chondr ocyt e differ enti ation b ut inde pe ndentl y of it to con trol proliferat ion. PTHrP neg ativ ely re gula tes c hondr ocyt e different iatio n: mic e with n o PTHrP ha d abnorm ally rap id

9 different iatio n of the prol iferat ing c ho ndrocyt e s into hy pertrop hic chondr ocyt e s, which cau se d dwarfi sm due to th e cho ndroc yte s’ reduc ed prolif erati ve tim e (Kara pli s, Luz et al. 19 94). The fibrobla st gro wth fa ctor s (FGF s) a re anoth er su bs et of protein s in th e EC M of cartil ag e. This 23-memb er family u s es a t lea st four rec eptor s (FGF R-1 to 4) an d splic e vari ant s of ea ch to regula te bon e de velo pmen t (Ornitz and Marie 2 002). FGF-2, 9, and 18 and FGF R-1, 2 an d 3 are all expr es s ed in the gr owth pl ate or it s surroun ding p eric hondri um. FGF-2 de c reas e s cho ndroc yte proliferat ion, hyp ertroph y and m atrix s ynthe si s (M anci lla, De Lu ca et al. 1998). FGF-2 n ull mic e, how ever , have no sev ere lon g bon e growth ph enot ype, show ing onl y mild adult on s et os teop eni a and som e redu ced bo ne d en sity (M onter o, Okad a et al. 20 00). Intere stin gly, mic e with a t arget ed ov e r-expre ss ion of FGF-2 o n growth pl ate c hondr ocyt e s are dw arfs ( Coffin, Florki ewi cz et al. 1995). FGF-2 i s mad e by growt h plat e chondr ocyt e s (Luan, Praul et al. 1996) an d sig nal s prim arily thro ug h FGFR-1 in c artil age. (W e have stud ied p erle can-m edi ated F GF-2 sign alin g throug h FGFR-1 and -3 in our re se arch. Our fin ding s ar e reporte d in Pap er II of this dis sert ation). FGF-9 i s al so m ade b y g rowth pla te ch ondro cyt es where it stim ulat es gro wth pl ate chon d rocyte pr olifer ation a nd inhibit s termi nal diff erent iati on (ie. di fferenti ation of pro liferat ive into hyp ertrophi c c ell s). Mic e over- ex pres sin g FGF-9 are al so dwarv es, ex hibit ing in crea s ed cho ndro cyte pro lifera tion bu t none of the differ enti ation n ec es sar y to form t he matrix sc affold for ne w bone (G arofalo, Kl iger-Sp atz et al. 19 99). FGF-18 is e xpre s sed i n perich ondriu m and i s kno wn to s ign al t hrough FGF R-3 to stimul ate chondr ocyt e prolif eratio n (Dav ids on, Blan c et al. 20 05). FGF-18 null mic e hav e redu ced pr olifer ation o f their growth p lat e chondr ocyt e s, which r esu lts i n sh ort b one s. They al so e xhibit

10 ribcag e deform itie s, del aye d os sifi cati on of endo chon dral bo ne s an d reduc ed tran sit ion of prolif erati ve ch o ndrocyt e s to a hyp ertroph ic pheno type (Liu, Xu et al. 200 2; Ohb ay ash i, Shibay ama et al. 200 2; Liu, Lavine et a l. 2006). VEGF is a noth er import ant grow th fact or in the growt h plat e ECM. In cho ndroc yte h ypertro phy, the ECM aro und hy pertrop hic cell s is i ncre as ingl y cal cifie d, allow in g blood v es se ls to in vad e the growth pl ate from th e und erlyin g met a phys eal b one. VEGF i s expre s sed in t he se hy pertro phic chon drocyt es, wh ere it prom ote s new bo ne format ion by pro motin g bloo d ves s el inva si on and t hu s coupl es v a scul oge ne si s to os teog en esi s to regulat e long b one gr owth (Gerber, Vu e t al. 1999). Integ rin s: The inte grin fami ly of cel l surfa ce re cept ors i s resp ons ible for m aint ainin g commu nic ation b etw een th e EC M and the cho ndro cyte ( Ruo sla hti 199 1). The proper co mmuni cati on betw een th e EC M and th e ch ondro cyt e lead s to prop er ce ll atta chme nt, differe ntiat ion, growt h an d surviv al (Loe ser 20 00). Chon drocy te s expr es s v ariou s membr a ne inte grin s that bi nd to EC M molec ule s s uch a s fibro ne ctin (whi ch a ffect s cell adh esi on), colla gen s (whi ch aff ect c ell an d matrix struct ure), lamin ins (w hic h control cell sha pe an d motili ty) and v i tronec tin (whi ch co ntrol s ce ll migratio n and sign al tran sd ucti on). It is cle ar, then, ho w indis pen s able in tegri ns ar e a s link s bet ween t he ch ondro cyt e and it s ECM. With mi sex pre ss ed int egrin, mam mmal s exp erie nce m ultipl e level s of gro wth dy spl asi a. For exa mpl e, los s of ß1 int egrin (whi ch is norm ally e xpre ss ed in a ll growt h pla te chon droc yte s) ca us es dwarfi sm in mic e re sulti ng from ab se n ce of the n ormall y orderly column ar arran gem ent of ch ondro cyt es due to redu ced m otilit y of

11 chondr ocyt e s, perturb ed cho ndro cyte morpholo gy an d redu ced chondr ocyt e prolif eratio n (Be ngt ss on, Asz odi et al. 2005). Matrix met allop rotein as es ( MMPs): The M MPs are the enzy me s that d egra de an d remod el th e cartil age EC M. In doin g so, they prov ide tur nover of EC M mol ecu l es, ther eby m aint ainin g the integrit y of the c artila ge EC M an d of the cho ndroc yte s. M MP s enab le ne o-va sc ulari zati on of the gro w th plat e (Vu, Shipl ey et al. 1998). This a ngio gen es is i s clo s ely co upled to b one form atio n in the hypertro phic zon e as d e scrib ed ab ove. A murine kn ock out mod el for MMP- 9 sh ow s abn ormal bo ne forma tio n as a re sul t of redu ced growth pl ate v as cul ariz ation ( Rath, Hu ff et al. 1997; Vu, Shi pley et al. 1998). M MP-13 i s al so kn own to b e nece s sary for ma trix mineral iza tion in th e hyp ertroph ic z on e by degr adin g coll age n 2 in the prolif erativ e zo ne an d promoti ng t he expr es sio n of coll age n X (D'Ang elo, Ya n et al. 20 00). The M MP s, in degr ading t he EC M, al so relea se l oca l growt h factor s (or s yst em ic one s th at hav e diffu sed i n) that are b ound b y EC M mole cule s so t hes e fac tors can aff ect chondr ocyt e ac tivity o nly wh en n eed ed and only in t heir s pe cific growth pl ate z one s.

How Lon gitudinal Bone G rowth O ccu rs. The growth pl ate E CM mo lec ule s di scu ss ed a bove all contrib ute to th e growt h of long b one s and oth er bon es th at re sult from a cartil age pr ecur sor. It is th e co mplex int erpl ay of the se molec ule s tha t lea ds to a) chon droc yte proliferati on b) cho ndro cyte hypertro phy a nd enh an ced EC M prod uc tion and c) chon droc yte apopt osi s th at re sult s in bon e growt h.

12 2a. Proliferati on The prolifer ation of c hondr ocyt e s prod uce s bon e growt h. Only the cho ndro cyte s of th e prolifer ating z one of the gr owth pl ate activ ely re plic ate, wit h ea ch c ell in thi s zon e produ cin g up to 8 ne w clon es e ac h day duri ng rapi d growt h p has e s su ch a s pub erty (Farn um and Wil sm an 199 3). Locally synt he siz ed PTHrP, Ihh and TGF-ß a ct in a car efully c oordin ate d feed ba ck lo op to contr ol whe n proliferat ive cell s be gin to term inal ly different iat e into hypertro phic cell s. The se prot ein s thu s control th e leng th of time for which e ac h cell exhib its pr olifer ati ve ca pac ity (B allo ck an d O'Ke efe 20 03). FGFR-3, whi ch bin ds at le ast 9 diff ere nt FGFs, i s a neg ativ e regula tor of cho ndroc yte prol iferat ion (Deng, Wy ns haw- Bori s et al. 1996). Whe n di srupt ed in mic e the re s ult is a bnorm ally prol onge d proliferat ion a nd thu s ov ergrowt h of lo ng bone s (C olvin, B ohn e et al. 1996). Con ver sely, a ctiv ating m uta tions of t he FGF R-3 ge ne cau se a cho ndropl a sia. In thi s mo st com mon of huma n dwarfi sm s (1:15, 000 to 1 :40,000 b irth s), FGFR-3 inhibit s ch ondro cyt e proliferat ion, whi ch, in turn, le ad s to s evere b one short enin g (Shiang, Thomp so n et al. 199 4). With t he Ihh/PTHrP/TGF- ß loop unden iabl y cruc ial in c hondr ocyt e prol iferatio n, it is an ticip at ed then th at FGF Rs a nd thu s FGF s c an mo dulat e thi s path way. Ther e is evid enc e for this i n the repr es si on of Ihh with FGF R-3 ov er- expre s sion (N as ki, Col vin et a l. 1998). The FGFR-3-re lat ed hum an chondr ody spl asi a s are sev ere a chon dro pla sia, mild er hypoc hondr opla si a, se vere (oft en let ha l) thanat ophori c dy spl asi a and SAD DAN ( se vere a cho ndropl a sia with de velo pmen tal d elay and aca ntho si s nigri can s) (Shia ng, Thomp s on et al. 199 4; Tavormi na, Rimoin et al. 199 5; Na ski, C olvin et al . 1998). These form s of dwarfi sm all re sul t from the a ctio n of FGFR-3 a s a n ega tive

13 regula tor of growth p lat e cho ndroc yte proliferat ion. The p erlec an disrup tion in mi ce a nd hum an s al so c a use s mar kedl y redu ced proliferat ion of gro wth pla te ch ondro c yte s, most li kely b y affec ting the av aila bility of F GFs to F GFR-3. F GFR-1 al so h a s role s in bon e growth a s s ee n in the d ysp la sia th at re sult s if thi s rec eptor i s mi s- expre s sed ( Mue nke a nd Sch ell 19 95; W hite, Ca bral e t al. 2005). Human gr owth h ormone and IGF-1 are als o know n to incr ea se growth pl ate c hondr ocyt e prolif eratio n (Ohls son, Is gaar d et al. 1993). IGF-1 may als o enh anc e the sti mulatory acti vity of TGF-ß on growth pl ate c hondr ocyt e prolif eratio n (O'Ke efe, Cra bb et al. 19 94). Modu lati on of the c ell c ycl e is th e hyp othe siz ed me cha ni sm for the compl ex int egrati on of the s e mole cul es that affe cts pr olifer ation in the growt h plat e. Pho sphory lati on of h iston e s and of prot ein s s uch as R b (retino bla sto ma prot ein) i s contr olled by cycl in de pend ent kina se s (C DK s). With a ctiva tion of CD Ks, the cell c ycl e prote in s suc h a s hist one s a nd tran scri ption f act ors are mo difie d (su ch a s by acet ylat ion) an d growth p lat e cel ls m ai ntain prol iferat ion. Different iati on of re sting c hondr ocyt e s to proliferat ing chondr ocyt e s reple ni she s the pr olifer at ive zon e.

2b. Hypertro phy/In cre as ed Matrix Pro duct i on Mat ure prolif eratin g cel ls diff erent iat e to a hypertro phic st ate. The chon drocy te s in the gr owth pl ate a re programm ed (an d al so influen ced b y loc al growt h fact ors a nd ECM mol ecul es) t o different iat e into ma ture hy pertrop hic chondr ocyt e s. This different iatio n is, th erefore, spon tan eo us if no inh ibitor s are expre s sed (V olk an d Leboy 1 999). B M Ps, for exam ple, ind uce maturat ion of th e prolifer ating chon drocyt e to a hyp ertroph ic s tat e. This mat uratio n bring s wit h it a s witch not only to th e larg er, rounder sha pe of th e hypertr ophi c cel l, but als o to the synt he si s of

14 marker s of the hy pertro phic cell ty pe, suc h a s coll age n X (Kronen berg 20 03). The incr ea se in cel l siz e is no t sim ply by a diffusi on of wat er into th e ce ll but it i s al so vi a an a ctiv e incre a se in the num ber s and siz es of or gan elle s and va cuol e s withi n the different iatin g cel l (Bal loc k and O 'Ke e fe 2003). The ch ondro cyt es underg o up to a 10-fo ld incr ea se in i nt racell ular vo lume and up to a 2. 7-fold incr ea se in EC M vo lume (H un ziker, Sch enk e t al. 1987) acc ompa nied b y up to a 5-fol d incr ea se in cell met abo lic a ctivi ty a s mea sure d by incr ea se d protei n produ ct ion (Hun ziker, K apfing er et al. 1999). With t he se reg ulat ed in crea s es, ch ondro cyte h ypertr ophy and it s con comit ant e nha nce d matrix s ynthe si s ac cou nt for up to 60% of long bon e growt h in en doch ond ral bone s th at grow ra pidly, suc h a s the hum eru s and f emur (Farnu m and Wil sm an 198 9). Hypertro phy no t only c ontrib ute s to gr owth pl ate-m edia ted longitu dina l growth m orphol ogic ally b ut als o met aboli call y. By synt he sizi ng incr ea se d coll age n X an d alkal ine ph os phat a se (AP), the EC M of hyper trophi c cel ls i s prep a red for cal cific ation (Ander son, H su et al. 1997). AP i s pre sen t in cho ndroc yte v esi cl es se crete d by th e hypertr ophi c cel ls in to the matrix. The se m atrix ves icl es c onta in c alciu m and AP and in itiate mi nera liz ation. C olX can bin d to the s e ve sicl e s and se cure t hem in th e ECM, thu s supp orting d epo siti on of the cal cium-l aden v e sicl es and th eir carg o into the EC M (An der son, H su et a l. 19 97). The matrix v esi cle s al so cont ain M MP s, whic h degr ade t he ex is ting EC M and rem ode l it into a latti ce th at ha s incr ea se d hydrox yap a tite cry sta ls a nd oth er materi als cruci al for maki ng bon e. The calcifi ed c artila ge c an th en serv e a s a temp lat e for bone form atio n .

15 2c. Apopto si s With th e ongoi ng prolif erati on and d ifferenti ation of gro wth plate cell s, a fina l pha se of re gula tion occur s wit h the rem oval of the mat ured c ell s that h av e se cret ed th e matrix pr ecur sor tha t ost eobl ast s wi ll us e for bon e formatio n . This remov al is by a pre- programm ed c ell de ath or ap opto si s. This i s the fat e of growt h plat e chondr ocyt e s that h ave t ermin ally diff erenti ate d into hy pertrop hic chondr ocyt e s and h ave se cret ed the m a trix sc affold for bo ne formation (H atori, Kl atte et al. 199 5; Shapiro, Ad am s et al. 20 05). After ca lcifi cati on of the m atrix aro un d the se hyp ertrop hic c ell s, apopt osi s a nd cl earin g of the se c ell s o ccur s. Mat ure hy pertrop hic cell s tha t are re ady to un derg o apop to s is s how morh phol ogic al chan ge s that are s imilar to o ther c ell s underg oing a popto so si s s uch as c ell s hrinki ng, DNA fra gme ntati on and pla sm a membr ane disrup tion (H atori, Kl atte e t al. 1995). The sam e produ ctio n of phos pha ta se s that pr omot es mi neral iza tion of the EC M al so pro duc e s phos pha te ion s th at indu ce th e apo ptot ic cha nge s in th e mo st matur e of the hyp ertrophi c zo ne c ell s. FGF-2 al so bind s FGF R-3 to c au se a po ptosi s of gro wth pl ate chondr ocyt e s (Sahni, R az e t al. 2001). This migh t expl ain w hy ther e is incr ea se d and pr emat ure ap opto si s a nd redu ced bo ne gro wth in mice ov er-ex pre ssi ng FGF-2 ( Coffin, F lorkiew icz et al. 199 5). Perlec an mig ht further c ontrol th is apo ptosi s by re gul ating th e avail abili ty of thi s FGF. Chon drocy te ap opto si s is n ec es s ary for growth-pl ate me diat ed bone form ation. Wi th their d eat h, the c hondro cyte s le av e the hypertro phic zon e open for in va sion of vas cula ture from th e adja cen t meta phy se al bon e. With th e se new bloo d ve ss el s com e enzy me s and o st eoc yte s tha t further de grade th e min erali zed m atrix and remo del th e cart ilag e (the o st eocl a st s) and th at al so la y dow n

16 the ne w bon e (the o ste obla st s). This li nk betw ee n ost eog ene si s an d vas culo gen e sis i s co ntroll ed by v as cul ar endot heli al grow th fact or (VEGF) (Gerber, Vu et al. 1999). VEG F is, predi ctabl y, expre s se d in hypertro phi c chon droc yte s but no t in resti ng or prolifer atin g chondr ocyt e s. VEGF rec eptor (VEGF R ) is al so only expre s sed i n the hypertro phic cell s of th e growth p lat e. FGF-2 is al so kn own to promote angio gen es is a nd s ub seq uent os sific ation of cartil age. Perlec an c an not o nly reg ulat e the a vai labilit y of FGF-2 but a ls o ha s well-re porte d angi ogen ic prop ertie s c o nferred by it s C-t ermin al domain ( Mon giat, Sw een ey et a l. 2003 ). FGF-18 is al so kn own to incre as e expr es si on of VEGF an d to m odulat e it s angi oge nic eff ect s (Liu, Xu et al. 2002). Expre s sion of F GF-2, FGF-18 an d perle ca n in the hyp ertroph ic zo ne mig ht further c o ordinat e the v as cul ariz ation that i s an e s sent ial e ndpoi nt in en doc h ondral bo ne form ation.

Perle can 4a. Structure /Fun ction Perlec an w as fir st ide ntifie d a s a HSP G in murine b as eme nt membra ne s in 198 0 (Ha ss ell, Ro bey et al. 1980) an d ha s si nce b ee n sho wn to be in chic ken s, flie s, worm s as w ell a s oth er mamm als (Roga ls ki, Willi ams et al. 199 3; Sund a rraj, Fite et al. 19 98; Voi gt, Pflanz e t al. 2002). R otary sha dow ed i mage s of murin e perl eca n sho w that th e cor e protei n of perle ca n, with its gl obul ar doma in s spa ce d by rod-lik e s equ enc es, re sem bl es a string of p earl s, hen ce it s name (No ona n, Fulle et al. 1991). The over 120 k b of the perl ec an gene enco de a pro tein core th at c an ex cee d 460 kD a in ma s s (for exam ple, in hum an an d mic e). The perl eca n core i s mod ular, con sis ting of fiv e doma in s that sho w h omology t o vario us

17 molec ule s. CS an d HS GA Gs are att ach ed at th e first a nd la st of the se dom ain s (dom ain s I and V; Fig ur e 2*).

Figure 2: Stru cture of p erlec an, s howi ng its 5 do main s a nd the atta che d GAG s. *From Smit h S. M-L. and Ha ss ell J. R. , Experim ental Eye Re se arch 8 3 (2006) 47 1-2.

The syn the si s of perl eca n beg ins as all protein s do wit h the trans cripti on of the core prot ein m RN A from the ge ne s equ enc e. The mRNA i s th en tran sl ated i nto a pre cur s or protein a nd c ertain intitia tion site s (Se r in Fig ure 3) on th e precur sor ar e us ed to initiat e CS a nd/or HS synt he si s (Yan ag ishit a and Ha sc all 199 2). This initi atio n is vi a add ition of x ylo s e (Xyl) in st ep 2 to spe cific serin e re sidu es - th at is, serin e res idu e s follow ed by a glyc ine a nd, in som e ca se s a n aci dic am ino a cid (an asp artic a cid or glu tami c acid). Ste p 3 is th e tran sfer of two g al acto se (G al) re sidu es t o the Xyl. Next, th ere i s addi tion of a gl ucur onic a cid re sidu e (Glc A) to compl ete thi s t etra sa cch aride li nka ge r egion ( step 4). At ste p 5, the synt he si s of CS an d HS div erge; N- ac e tylgal act os amin e (Gal NAc) i s adde d by Ga lNA c tran sfer as es t o the li nkag e regio n in CS synth e sis while N- ac etylg luco s amin e (GlcN Ac) i s add ed by EXT1 a nd 2 in HS synt he si s; a clu st er of aci dic re sid ue s within ~ 7 resi due s of th e 18 serin e favor s HS synt he si s on that seri ne. Additi on of rep eat ed altern ating Glc NAc and Gl cA re sidu e s then serv e to polym eriz e the HS chai n (st ep 6). Mod ifica tion of th e growing H S cha in (st ep 7) by N-dea cet ylati on, N-s ulfati on, epim eriz ation a nd furth er sulf ation (2- O, 6-O and, rarel y, 3-O) yield s th e full y modified GAG a nd, thu s, the com plet e prote ogly can. Epim eriz at ion of Glc A to iduron ic a cid als o occ urs for CS, wi th the t erm CS if GlcA ge ts in corp orate d and “derm atan sulf ate ” or DS if IdoA i s pres ent (Ha s call, C ala bro et al. 1994).

Figure 3: Bi os ynth es is of p erle can. Th e first s tep of p erle can synt he si s is th e tran sla tion of th e core protein w ith it s s erine G AG atta chme nt si te (Se r) an d the gl ycin e (Gly) that fol low s it. The se cond thr ough sixt h st ep s (number ed 2 - 6) are indic ate d by arrow s. Step 7 invo lve s mult iple m odific atio ns such as a cet ylat ion, epimeri zat ion a nd su lfatio n (See t ext). *-first st ep wh ere CS and HS differ; A is G alN Ac for CS and Gl cNA c for HS and i s add ed by th e corre spon ding tra nsf era se s (for HS, th e tran sfera se s ar e sp ecifi cal ly termed EXT1 a nd 2 (He cht, Ha ll et al. 2002)). B is ur onic acid (for CS, glucur onic acid addit ion prod uc es CS, epim eriza tion to id uroni c acid pro duc es DS).

It is import ant to reit erat e tha t addit io n of CS ver su s HS is determi ned b y the am ino a cid sequ en ce in the prote in cor e. For inst anc e, if a seri ne ini tiati on si te in p erlec an i s follow ed by a glycin e, then GAG a tta chme nt will b eg in at that seri ne, but HS w ill only be a tta che d if the s erin e is fla nke d by a group of a cidi c amin o acid s s uch as a sp artic acid (D olan, H orchar et al. 1997). There are three suc h aci d-flank ed serin es i n the f irst dom ain of perl ec an an d

19 one in Do main V. The y are a ll Serin e- Glyci ne-A sp artic a cid (SG D) seq uen ce s flan ked b y aci dic re sid ue s (ie. –S-G-D-X-X-X- D/E-D/E- X) that fav or HS atta chm ent bu t can al so rec eiv e CS. There i s al so a spe cific CS an d an HS at tac hme nt sit e in domai n V (Tapana de chop one, H as se ll et al. 19 99 ).

Domai n I: This ~17 2 amin o aci d doma i n was i nitia lly co ns ider ed the only uniq ue do main of p erle can (N oon an, Fulle e t al. 1991). Howe ver an SEA h omolo gy dom ain ha s sinc e be en ide ntifie d that i s found in oth er prote in s and mig ht hel p to modulat e GA G att achm ent (Bork an d Patth y 1995). Exc ept for the three co ns ecu tive S GD HS atta chme nt si te s, there i s no rep eat ing struc ture in d omain I, whi ch disti ngui sh es it from th e oth er four per leca n prote in dom ain s. The GAG ch ain s att ac hed to d omai n I cau s e this d omai n to pla y a cruc ial role in bind ing c ertai n growth f actor s. The GAG s on do main I ha ve b een sho w n to bind to angio poie tin-3 an d PDGF (to aff ect ti s sue angio gen e sis), co llag en IV (to control cell m atrix s truct ure) an d FGF-2 (Iozzo, Coh en et al. 1994; Go vindr aj, We st et al. 2006; Smi th, Wes t et al. 200 6). In bindin g FGF-2, a growt h fact or with pl eiotropi c effe ct s in deve lopm ent an d cel l surv ival, p erlec a n help s to mod ulat e the actio ns of th is gro wth fa ctor, e spe cial l y its effe ct s on c artila ge an d endo chon dral bo ne grow th. FGF-2 will be de scrib ed in d etail l ater. The GAG s on do main I of perl ec an al s o bind fibron ecti n to co ntrol cell at tac hme nt and l amini n-1 to co ntr ol cell struc ture (Ioz zo 19 94). The prolin e/argi nine-ri ch e nd leu cin e-rich rep eat prot ein (P RELP) can al so b ind perl ec an’ s doma in I GA Gs to stab iliz e the cell by anch oring c ell s to the ir underl ying c on necti ve ti s sue (B engt s son, Morg elin et al. 2002). The GA G s on d omain I of perl ec an al so bin d

20 to VEGF (allo wing p erle can a rol e in c ontrollin g angi oge ne si s in variou s ti ss ue typ e s inclu ding t he c arti lage pr ecur sor in endo chon dral bo ne grow th) and t o thro mbos pondi n (su gge stin g a role for perle ca n in bloo d clott ing an d tis sue re mod eling) (Ioz zo 2005). The GA Gs of p erle can a ls o bind the amyl oid-ß prot ein s involv ed in am yloid oge ne si s and Al zh e imer’s di se a se to bo th acc eler ate a nd mai ntai n path oge nic fib ril formation ( Ca still o, Ngo et al. 1997).

Domai n II: This ~21 1 amin o aci d doma in is rich i n cy stei ne s, unlik e domain I wh ich h as n one, wh ich prom o te ext en sive t ertiary struc ture. The dom ain sho ws sub st anti al homol ogy to th at regi on of the LDL- rece ptor tha t bind s lipi ds, wi th four ~40 ami no a cid LDLR-like re pe ats. Wi th it s homol ogy to LDLR, Domai n II has so far been sho wn to int era ct with VL DL. A DGSDE amin o aci d seq uen ce pr es ent in ma mmali an p erle c an doma in II is kno wn to media te th e bindi ng of LDLR to LDL (Iozzo 2 005) an d might th us help to re gulat e lipo met aboli sm. Dom a in II may al so be a b indin g site for pro tein s wit h lipid mo ieti es. O ne su ch prot ein i s Ihh, whi ch has a chol e sterol m oiety atta che d to it and i s es s enti al for endo chon dral bo ne grow th (St-J acq ue s, Hammer sc hmidt e t al. 1999). In binding Ih h and e ither seq ue steri ng it from or deliv ering it to it s recep tor, domai n II and perl ec an wo ul d serv e ano ther vit al role i n chondr ocyt e ac tivity. Fol lowi ng the L DLR su bdom ain th ere i s a region of ~1 00 ami no ac id s cont aini ng a short re pe at homo logou s to N-CA M of the im muno globu lin ge ne s u perfamil y (Co stel l, Sas aki et al. 1996)

Domai n III: This larg e ~ 1170 am ino acid do main i s ma de up of three subd omai ns (IIIa to IIIc). Each o f the three sub doma ins of

21 domain III co nsi st s of a s ingl e cy ste in e-rich rep eat fol low ed by a cys tein e-free g lobul ar regio n then t hre e more cy st eine-ri ch rep eat s. The cy stei ne rich r epe at s are ~3 5% ho mologou s to re pe ats fo und in domain s III and V of th e lami nin A sh ort chai ns. The gl obul ar region s of the sub doma in are ~ 30% ho mologou s to d omai n IV of the laminin A sh ort cha in s (Noon an, Fulle et al. 1991 ; Cou chm an, Ljubimov e t al. 1995). Wit h the e xten s ive terti ary str uctur e that t he cys tein es pr omot e, domai n III seem s a good c andid ate for mu ltipl e intera ction s. Ind eed, dom ain III bind s to FGF-7 and F GF-BP to modula te gro wth-fa ctor med iat ed ce ll proliferat ion a nd PDGF to potent iall y contr ol angi oge ne si s (Iozz o , Cohen e t al. 1994). M urine domain III co ntai ns a n RG D s equ enc e (abs ent in hu man p erle can) that i s know n to medi ate t he bin ding o f integrin s to perl ec an an d, thus, to m ediat e c ell ad he sion a nd mot ility (Ch akra varti, Hor char et al. 1995).

Domai n IV: This i s the l arge st of th e p erlec an dom ain s. In huma ns, it cont ain s over 20 00 ami no ac id re sid ues t hat am ount to o ver 220 kDa in mol ec ular ma s s. Doma in IV is a serie s of rep eat s wit h homolog y to the im muno globu lin (Ig) gene fa mily, e spe cia lly N- CA M (Noon an, Full e et al. 199 1; M urd och, Dod ge et a l. 1992). There is als o a put ativ e GAG atta chm e nt sit e in the fourt ee nth Ig repea t of huma n doma in IV whi ch i s a bse nt in mic e, proba bly du e to altern ativ e sp licin g. Huma ns h ave a to tal of twe nty-on e of the se Ig repea ts v ers us th e fourte en in org ani s ms s uch a s mi ce an d nemat ode s. The tw enty-o ne Ig rep eat s in human d omai n IV are th e large st num ber of Ig rep eat s ide ntifie d so far in a ge ne pro duct (Murdo ch, Dodg e et a l. 1992). With s uch a defin ed ye t compl ex struct ure, it is no surpri se that dom ain IV bi nds m ole cule s a s div erse as coll agen IV,

22 fibrone ctin, fibuli n-2 (to regu lat e cell sha pe a nd mov emen t), nidog en (to co nne ct coll ag en an d the l aminin: nido gen c ompl ex) and PDGF (Iozz o 1994 ; Iozz o 2005). Perl e can c an th erefore, vi a its domain IV, re gulat e proc e ss es su ch a s growth fa ctor a ctivit y, angio gen es is, c ell ad he sion, c ell mobi l ity and c ell surviv al.

Domai n V: This ~ 705 a mino a cid do ma in is th e C-term inal re gion of perle can. Ag ain, th e stru cture i s on e o f three glob ular (cy st ein e- free) su bdom ain s link ed by c yst ein e-ri ch s egme nts. The g lobul ar subd omai ns (V a to Vc) sho w more th an 30% homolog y to lami nin A, even fol ding in to glob ular lo ops t he w ay lami nin do es. The fir st two globul ar doma ins are e ach fol low ed by two cy stei ne-ric h repe at s that s how ho molog y to EGF. The fourt h EGF repea t is th en follo wed by the C-t ermin us of p erlec an. In the s econ d and fourt h EGF repea ts, a Ser ine-G lyci ne (SGx G) s equ enc e occ urs, wh ich i s a putati ve GA G att ach ment sit e. Perlec a n thus can a cc ept tw o GAG chai ns on it s C-termin al en d doma in (Noon an, Fulle e t al. 1991 ; Murdo ch, Dod ge et al. 1992). Domai n V ha s be en s hown t o bind to e ndos tati n (the C- termina l end of c olla gen X VIII), ECM- 1, FGF-7, fibulin-2, inte grin s and nid ogen (Io zzo 2 005). In thi s way, perlec an c an affe ct pro ce ss es as di ver se a s grow th fact or acti vity, c e ll-cell and c ell-ma trix intera ction, c ell mot ility a nd ba se ment membran e st abili ty. Doma in V als o bind s α-dy strogly ca n at the n eu romus cular j uncti on in ske let al mu scl e to medi ate it s int era cti on with a cet ylch olin e est era se, th ereby re gula ting th e s ynap s e and fa cilit ating pr oper mus cle c ontra ction (P eng, Ali e t al. 19 98). Domai n V by it self h as b een sho wn to have bi olog ica l acti vity as “ end orep ellin ”, a mole cul e (name d t o allud e to its anti- angio geni c prop ertie s) tha t bind s en do stat in to fun ction in b a sem ent

23 membra ne a s semb ly (Mo ngia t, Swee ne y et al. 2003). The local iza tion of e ndore pelli n in the BM might al so b etter po sit ion th e GAG ch ain s of dom ain V to bi nd grow t h factor s and m aint ain B M functio ns in p erme abili ty an d traffick i ng. Mo st of the a ngio st atic activ ity of en dorep ellin se em s to re sid e in the C- termin al-mo st globul ar doma in, whic h ca n be lib erat e d from the re st of the c ore by protea s es.

4b. Gene and Muta tion s The protei n core of p erle can i s en cod e d by a si ngle ~ 120 kb gene w ith 97 e xon s (Noo nan, Full e et a l. 1991). The gen e (huma n chromo som e 1, mou se chr omo some 4) i s wel l con serv ed a cro ss spe cie s, with h omolo gue s of mam mali a n perle can (h spg 2 gen e) pres ent in C. eleg an s (unc-5 2 gne), D. melan oga st er (trol g ene) and other s pe cie s. The promot er of the p erl eca n gen e cont ain s TGF-ß resp ons e el emen ts th at mod ulat e the ac tivatin g effe ct of TGF-ß on perle can g ene tr an script ion (Ioz zo, Pil laris etti et al. 1997). Perlec an show s v ariety at man y lev els. In human s, the ge ne enco de s an ~4 66 kD a cor e protei n. Mic e can a ls o expr es s thi s form of perle can re sult ing from al tern ativ e spli cing, but t heir u sua l perle can g ene pr oduc t is a n ~369 k Da core prot ein (No ona n and Has s ell 199 3). Perlec an i s terme d a he paran sulfa te prot eogl yca n (HSPG), but in fac t the c ore prot ein c a n als o hav e CS GA G s atta che d. The varyin g su lfatio n patt ern s and G AG ty pe s of perl eca n in differen t tis su es pro vid es a noth er le vel of the in here nt vari ety of perle can. For ex ampl e, growth p lat e pe rleca n ha s both CS and HS (Govindr aj, We st et al. 2002; We st, Go vindraj et al. 2006) w her ea s EHS perle can (from the Eng elbr eth-H olm-Swarm tu mor) rarely h a s CS, but ha s HS (C ouch man, K apoor e t al. 1996).

24 Disru ption s in th e perl ec an gen e in dif ferent org ani sm s hav e been stud ied a nd th e res ulting d is order s ch aract eriz ed. In C. eleg an s, perle can m utati on s produ ce d efect ive mu sc le de vel opme nt cau sing p aral ysi s a nd s pa stic ity tha t make s the m ovem ent of th e nemat ode s un coordi nat ed (he nce t he “u nc” n ame for th e gen e) (Roga ls ki, Willi ams et al. 199 3). In Drosop hila, p erlec an mut atio ns cau se d efec tive eye b as eme nt mem bran e formatio n, leadi ng to abnorm ally small o ptic l obe s (he nce t h e name “tr ol” for th e gen e, for terribly red uce d opti c lobe s, on e of the phen otyp e s that re sul ts from this p erle can d efec t) (Voigt, Pfla nz et al. 20 02). Mut ation s ha ve al so b een char act eriz e d in the murin e and human p erle can g ene s. In 199 9, Arika wa-Hir as awa et al a nd Co st ell et al co ncurre ntly bu t s epar ately g en er ated mi ce wi th a mut ation i n exon 7 of th e perl eca n gen e (Arika wa- Hira saw a, Wat ana be et al. 1999; C os tell, Gu st afs son et al. 199 9). The resul ts w ere eff ecti ve knock out s of the p erle can g ene (h spg 2 ) and los s of perl ec an expre s sion in t he se a nimal s. Appr oxim ately 4 0% of the h spg2 n ull mice di ed at mi d-ge sta tion (E10.5). Th e remain ing nul l mic e died perina tally and show ed sev ere skel etal dyspl as ia in cludi ng microme lia, broa d and b owe d long b on es, narro w rib-c age, craniof aci al def ect s an d dwarfi sm (Fig ure 4*). Thes e re sult s est abli sh ed perl ec an a s a po siti ve reg u lator of cho ndro cyte proliferat ion a nd differ enti ation in t he growth pla te. A more via ble p erle can mut ant mo us e has als o be en gen erat ed where exon 3 of th e murin e gen e (whi c h code s for the GAG atta chme nt si te s in dom ain I) wa s del e ted (Ro s si, Morit a et a l. 2003). The se mic e s urviv e and ar e fert ile but th ey ha ve def ect ive eye s tha t are not o nly ~7 5% of the we i ght of normal eye s du e to incre as ed ap opto si s of len s ep ithe lial c ells, bu t al so hav e len s es which b ec ome am orpho us a nd ruptur e when th e mic e are ar ound 6

25 month s old. This m ou se mod el i s dis cu ss ed in sligh tly more d eta il in the Foc us Pa per in clud ed a s an a ppe nd ix in this d is sert atio n.

Figure 4: M orphol ogy of wil d-typ e (a, c, e) versu s h spg2 n ull (b, d, f) mice. Gros s a ppe aran ce (a, b) an d s kelet al pre parat ion s (c-f) sho wing d warfi sm re sultin g from defe c tive bon e grow th in endo chon dral bo ne s su ch a s th e fore li mbs (c, d) and h ind lim bs (e, f). *From Arikawa- Hira sa wa et al, Nat . Genet. 199 9 (23), 354-8.

Human d ys se gme ntal d syp la sia s ar e au tosom al re ce ss ive ske let al dy spla si as t hat c over a w ide r ange of sympt om s, all involvi ng dw arfism. D ys se gmen tal dy s pla sia Silv erma n-Ha ndma ker type (DDS H) is a se vere l eth al form of thes e dy spla si as. Clini cal and radi ologi cal simil ariti es a mong th e sk elet al dw arfism of D DSH patie nts and p erle can nu ll mic e prompt ed res ear cher s to e xamin e patie nts with D DSH to un cov er any mu tation s in th e perl ec an gen e. Dupli cati ons, po int mut ation s a nd exo n skip ping h ave a ll be en sub se que ntly id entifi ed in th e perle ca n gene of pa tien ts wi th DDS H and oth er dy spla si as (N icol e, Dav oine et al. 2000 ; Arika wa- Hira saw a, Wilc ox et a l. 2001; Ari kaw a -Hiras aw a, Wilc ox et al. 2001; H as s ell, Yam ada et al. 2002). Th es e mutat ion s ca us e vario us degre es of tru nca tion a nd de cre as ed fu nction alit y of the perl ec an 26 gene pr oduc t. In DDSH, thi s trunc atio n is s ever e eno ugh th at the trunca ted p erle can i s not se crete d by c ells from th e se p atien ts b ut is simpl y degr ade d intra cell ularly. The s e DDSH pat ient s ar e effecti vely p erle ca n null an d, like th e mous e mod el, die s hortly after birth. The d efec ts in endo cho ndra l bone gro wth in th es e patie nts h av e al so und ers core d the po s itive rol e of perle ca n in growth pl ate c hondr ocyt e prolif eratio n . In the mild er chon drody spl as ia kn own as Sc hwart z J amp el syndr ome (S JS), mis sen s e and spli cing mutation s re sult i n the product ion of a trun cat ed p erlec an pro duct th at is still se cret ed an d remain s part iall y functi onal. Pa tien ts s till suff er the dw arfis m and ske let al abn ormalit ie s of the D DSH p a tient s but on a much mi lder sc ale.

Fibrobla st Gro wth Factor (FGF)-2 5a Structure /Fun ction FGF-2 (ba sic FGF) i s on e of twen ty-th ree memb er s of the FGF family. FGF s rang e from 17-34 k Da an d have amino a cid re sid ue s that int erac t with FGF re ce ptor s (FGF Rs). FGF-2 i s ~18 k Da a nd als o cont ain s s ever al ba si c res idue s th at form a hep arin/ HS bindi ng site d ist inct from th e FGF R bindi ng s it e. FGF-2 doe s not c ont ain a sign al s equ enc e but i s s ecre ted b y som e unkno wn pa thw ay (Ornit z and Itoh 20 01). It is norm ally e xpre s se d at low le vel s but i s upregul ate d in pat holo gy or in regi ons of high ce ll act ivity suc h a s in tis sue r ege nerat ion an d bon e format ion. FGF-2 wa s origi nall y identifi ed in bo vin e pituit ary (Go spo d arowi cz 197 5) but wa s fir st purified from gro wth pl ate c hondr ocyt es (Sulli van and Kl ag sbrun 1985), wher e it affe ct s variou s c ell a ct ivitie s by bi nding t o the FGFR s expr es s ed on th e cho ndroc yte s urface. The FG FR th en dimeriz e s and a utop hos phory lat es sev e ral intra cellu lar tyro sin e

27 resid ue s. Thes e pho sp hotyro sin e re sidu es th en recr uit prot ein s that activ ate k nown pr olifer ativ e ca sc ade s suc h a s the MAP kin as e pathw ay in c ell s. Des pite b ein g mad e by div ers e cel l typ es, FGF-2 i s targ ete d sub sta ntia lly to th e extr ace llul ar matri x of cell s for stor age o n low- affinity re cep tors u ntil ne ed ed. Perle ca n is the l ow-affin ity rec ept or for FGF-2 and FGF-1 8 in the gr owth pl ate (refer enc e s: (Govi ndraj, We st et al. 20 06; Smith, W e st et al. 20 06) and Pa per II of this dis sert ation). Perl ec an c an al so fa cilit ate th e bindi ng of FGF-2 to its ch ondro cyt e rec eptor s FGF R-1 (pre sen t in prehy pertro phic chondr ocyt e s) and FGF R-3 (pre se nt in proliferat ing c hondro cyt es). Both th e FGF an d the FGF R ha ve h epa rin/HS bind ing site s, thu s supp orting th e ide a of a tern ary co mpl ex of FGF, FGFR and HSP G that form s to modul at e FGF si gnal ing ( Ornitz a nd Led er 1992; Ornitz, Xu e t al. 1996 ; Ornit z and Mar ie 2002).

5b. Mut ation s With it s plei otropi c loc aliz atio n and fu nction, FGF- 2 alter s diver se p athw ay s if mis- expre s sed. Mi ce with a targ eted d ele tion of FGF-2 hav e be en gen erat ed a nd st udie d (Dono, Texid o et al. 199 8; Orteg a, Ittmann e t al. 1998; Mon tero, Okad a et al. 20 00; Am ann, Faulh aber et al. 2006). The se nu ll mic e are vi able b ut suff er from reduc ed c ardia c ca pillar y formatio n (t hus lo werin g their a bility t o resp ond to c ardia c hyp oxia), d efect ive blood pre s sure r egul ation (which l ead s to hy pote ns ion), ineff ecti ve contr ol of va sc ular ton e and del ay ed wou nd he alin g. They al so exhibi t impair ed de vel opme nt of their ce ntral n ervou s s ys tem. M ost i mportant ly for our re sear ch intere st in FGF- 2 is th e ab se nce of m aj or defec ts in endo chon dral os sific ation i n the se FG F-2 defi cien t mice. There i s onl y a mild adult-o ns et o steo peni a (de crea s ed bon e mineral d en sity a nd bon e

28 formation). Thi s sli ght def ect i n ost eo gene si s oc curre d with no defec ts in chon droge ne si s. How ever, e arlier s tudi es sho wed th at when FGF- 2 is ov er-ex pre ss ed in a tr a nsg enic m ou se mod el, the resul t is a d warf mou se d ue to d ecre a s ed differe ntia tion of chondr ocyt e s from the prolif erati ve int o the hyp ertroph ic ph enot ype (Coffin, Florki ewi cz et a l. 1995). FGF -2 is thu s a ne gati ve reg ulat or of long bon e growt h. With th e simi lariti es of th e perl ec an n ull mou se to th es e FGF- 2 trans geni c s, FGF-2 be cam e an int ere sting fo cu s for our re sear ch in the lab. W e found pr evio usl y that F GF -2 binds t o perl eca n from the fetal bo vine rib gr owth pl ate (o ur mod el sy st em in the l ab) but i s not deli vere d to the FG FR (Go vindr aj, We st et al. 20 06) and h av e als o dis cov ered o ne of the r ea son s for this no n-del ivery. The se latter re sult s ar e pre sen ted in P aper I of this di s sert ation.

FGF-18 6a. Structure /Fun ction FGF-18 is approx imat ely 22 k Da in it s activ e, proce s sed form. FGF-18 is 9 9% con ser ved b etw een hu m ans and mi ce an d sh ow s mos t homolog y (>50%) to FGF- 8 and FGF- 1 7. FGF-18 is e xpre s sed in adult lu ng s, kidne y, liver an d sm all int esti ne in a sp atial and tempor al mann er di stin ct from oth er FGFs, s ugge sti ng th at FGF-18 has u niqu e role s in c ell a ctivit y (Ornit z 2005). In carti lag e, FGF-18 is ex pre ss ed onl y in the p erich ondri al layer aro und th e growt h plat e cartil age. Thi s expr es sio n patt ern s ugg est s th at FGF-18 i s a potent ial reg ulat or of long bo ne gro wt h. FGF-18 bind s pref erenti ally to FGFR-3 b ut ca n al so bind F GFR-2. FGF-18 al so c onta ins a hepari n-bind ing dom ain a nd i s in fact known to b ind to h epari n (Ornitz 20 05). In cartil age chon droge n esi s, FGF-18 stimu lat es

29 ost eobl ast s a nd cho ndro cyte s a s s trong ly as FGF- 2 doe s but i n an FGFR-3-m edia ted, he parin /HS ind epe n dent ma nner. FGF-1 8 is al so known to b ind to FGF R-2 to c ontrol o s teog ene si s and VEGF to control v as cul ogen e sis, bo th of whi ch are al so e s sent ial step s in endo chon dral bo ne grow th (Liu, Xu et al. 2002; O hbay a shi, Shibay ama et al. 2002 ; Liu, Lavine et al. 2006). In matur e bon e, FGF-18 ha s be en show n import ant for cartil age re pair (M oore, Ben del e et al. 200 5).

6b Mut ation s Sever al line s of mic e with a targ eted d isrupt ion in th eir FGF- 18 gen e hav e be en ind epe nden tly ge ner ated by d ifferen t group s. Liu et al (Liu, Xu et al. 20 02; Liu, Lavin e et al. 2006) g ener ate d an FGF-18 null allel e throu gh homo logo u s reco mbin ation, e limin ating exon 1 of th e codi ng regi on. Mi ce h ete rozygo us for th e null al lel e were ph enot ypic ally n ormal. Ho wev er, homozy gou s prog eny from the se norm al mut ant s, died wi thin thirt y minute s aft er birth. Their defec ts in clu ded b eing 10- 15% sm all er than wild t ype or hetero zyg ote litt ermat e s, more tha n 90 % having c left pa lat e, and respir atory fa ilure du e to thor aci c/rib c age d eformiti es. In th e null mutant mo us e, os sifi cati on wa s d elay e d by ~ 2 day s com pare d to normal litt ermat e s; thi s led to short ene d os sifie d region s in b one s and, ther efore, s horten ing a nd curv atur e of bone s s uch as th os e of the fore- an d hindl imb s. Co-o ccurrin g with the s e defe ct s in the appe ndic ular skel eton, ot her en doc hon dral bon es suc h a s the craniof aci al bon e s were als o abn ormal. The skull w as sma ller an d more round ed in FGF-1 8 null mi ce a nd the jaw bone wa s underd eve lop ed. At the hi stol ogic al le vel, th e zon es of proliferat ing c ell s in the FGF-18 null gro wth pl ate s w ere ~14% longer a t E16. 5. Similar

30 incre as es were se en in th e FGFR- 3 nul l mous e. How ever, for FGF- 18 null mic e, thi s incr ea se di sa ppe ars l ater in em bryog ene si s. In the FGFR-3 nu ll mic e (Colv in, Boh ne et al . 1996), thes e ex pand ed proliferat ive zone s re sult i n overgr owt h of long bon es b ut in the FGF-18 null it re su lts i n dwarfi sm sinc e the in crea se i s onl y trans ient. From thi s FGF- 18 null mo us e, lack of FGF-1 8 wa s dis cover ed to in hibit c hondr ocyt e proli feration for th e maj ority of embryog en esi s, le ading t o dwarfi sm. Liu et al (Liu, Lavin e et al. 2006) lat er di scov ered t hat a s e arly a s at E14. 5 of embryo gen e sis (when cr ani al os sifi cati on be gin s in wi ld type mi ce but n ot in th e null mic e and lim b formati on is alre ad y underw ay in bo th) the n ull mice a ctu ally h ave 26- 31% sh orter pro liferativ e an d hyp ertrophi c zone s. This i s in c ontra st to th e incr ea sed pr olifera tion a nd different iatio n of cho ndroc yte s at E16. 5 and in sign ifica ntly different pr olifera tion a t E18. 5 (Ohb ay ash i, Shibay ama et al. 200 2). A multiph a sic a ction of F GF-18 oc cur s , then, where t he s ame chondr ocyt e s at an org ani sm’ s differe n t devel opme ntal sta ge s resp ond differ entl y to FGF-18/F GFR- 3 sign aling. Earli er on (E14.5) this sign alin g in normal embryo s i s mi togeni c an d differe ntiat ive, leadi ng to norm ally hi gh produ ctio n of proliferati ve an d hypertro phic chon drocy te s. Later on ( E16.5), the FGF-18/F GFR-3 sign al is n ormall y anti- prolifer ativ e an d anti-diff erenti ativ e, reignin g in the c hondr ocyt e ac tivit y so proper hyp ertrophi c zo ne bone synth e sis can o ccur. A s embr yog e nes is c ontin ue s (E18. 5), the normal c hondro cyt es p artly re vert to b eing stimul ate d by FGF- 18/FGF R-3, cau si ng a ne wly in crea s ed pool of prolif eratin g cel ls and of differ enti ated h ypertro phi c cell s, enc ourag ing co ntinu ed endo chon dral gro wth. Unlik e the simil ar chon drocy te ph enot ype s s een in th e two mous e mod els, th e del aye d os sifi cati on in FGF-18 null mi ce w as n ot

31 se en in th e FGFR-3 n ull mic e. In contr ast to chon droge ne si s, then, FGF-18 do es no t sig nal thr ough FGF R- 3 to control o st eog ene si s. It is thou ght th at FGF-18 m ost li kely pr o mote s ost eob la st mat uratio n and prolif erati on throu gh FGF R-1 expr es se d in hyp ertrophi c chondr ocyt e s or FGFR-2 expre s sed i n the peri chon drium. Whil e regula ting c hondro gen e sis v ia FGF R-3, FGF-18 is th erefor e al so regula ting o ste oge ne si s via FGF R-1 a n d/or FGFR-2 and reg ulat ing vas culo gen e sis vi a VEGF a s d es cribe d earlier.

Histon e H3 7a. Structure /Fun ction Hist one s, like F GFs, ar e anot her s et of develo pme ntall y importan t protei ns. The y are hi ghly c a tionic pro tein s e s sen tial for bindin g and c ompa ctin g chrom atin in t he nucl eu s of mamm ali an cell s. The com pa ct natur e of the c hrom atin fiber s re stric ts th e intera ction of pr otei ns wi th DN A and r egul ate s gen e a ctivit y. The chroma tin co nsi st s of rep etitiv e unit s c alled n ucl eo some s, whi ch con sis t of an oc tam er of hist one s (two eac h of H2A, H2 B, H3 an d H4) aroun d whic h two sup erheli cal t urns of DN A are wr app ed. This core part icl e is th en follo we d by a DN A region th at lin ks a dja cen t nucl eos ome p articl e s (Schmi ede ke, Sto ckl et al. 19 89). Hi ston e H3 is ~19 k Da in siz e. Like a typ ica l his to ne, it ha s many b a sic resid ue s. H3, how ever, al so h as an Arg inine re pea t (RR xR) sequ en ce near it s C-t erminu s whi ch al ign s perfe ctly wit h the c on sen su s hepari n bindi ng s equ enc e YY xY wh ere Y= Argini ne or Lysi ne. Hist one s are i nde ed kno wn to bi nd to h eparin and HS (Sc hmie dek e, Stockl e t al. 1989 ; Wat son, G ooder ham et al. 1999; H enriq uez, Ca sar et al. 2002). This b indin g oc curs both in the n ucl eu s and extran ucl early. The extran ucl ear lo cali zatio n of his tone s h as b een

32 sho wn wid ely (Sch mied ek e, Stockl e t a l. 1989; Wa tso n, Edward s et al. 1995; H enriqu ez, C a sar et a l. 2002) .

7b. Mut ation s To date, no hi ston e kno cko ut muta nt s have b een gener ate d. With th e integr al role of th e hist one s in c hromat in struc ture a nd gen e a ctivit y, I would s p ecul ate th at co mple te lo ss of even a sin gle hi sto ne such as H 3 woul d be inco mpat ible with life. Hist one H 3 ha s multi ple re sid ue s that can b e pos t-tran sl ation ally modifie d by ac etyl, me thyl a nd pho sp h ate grou ps. Su ch coor dina ted modific ation of h ist one s co ntrol s chro matin struct ure an d the acc es si bility of tra ns cripti onal pr otei n s to the DNA, thu s reg ulati ng gene acti vity (W ats on, Good erha m et a l. 1999). For examp le, histo ne a cet ylati on i s cat aly zed by h ist one a cety l tran sfera s es (HATs) to rel ax chr omati n stru ctur e an d promote g ene tr an script ion when tr ans cripti on fac tors g ain a cc es s to the DN A. His ton e dea cety la se s (HD AC s) modify t he hi st one s to cou nterb ala nc e the activ atin g effe cts of H ATs. Sev eral H DAC s ha ve b een kn ock ed out in mous e mod el s, includ ing H DA C 4, 5 and 9. Only mi ce la ckin g HDA C4 ex hibit ske let al defe ct s (Veg a, Mat sud a et al. 20 04). The growth pl ate c hondr ocyt e s of the se H D AC4 nu ll mic e sh owe d premat ure hyp ertroph y, lead ing to pr e mature o s sific atio n and ecto pic forma tion of bo ne in norm ally cartil agino us elem ent s. The se defec ts affect ed th e muta nts’ vi abil ity by makin g it diffic ult to breath e, move and suck le. In fact, no HDA C4 null mi ce survi ved to wean ing. HD AC4 i s norm ally e xpre s se d in hypertro phi c chondr ocyt e s. The knoc kout mo del h as show n that H DA C4 norm ally bind s to Ru nx2 an d inhib its it s a ctivi t y. Runx 2 is th e only tr an script ion fa ctor proven n ec es sar y for chondr ocyt e hyp ertroph y and o st eobl as t differenti atio n (Inad a,

33 Yas ui et a l. 1999; Ki m, Otto et a l. 199 9). Runx2 mu st b e regul ate d during ch ondro cyt e hyp ertrophy so th a t prematur e and eve n ect opic bone form ation d oe s not o ccur. HD AC 4 negat ivel y regul ate s R unx2. With HD AC 4 mis sin g in thi s mou se mo del, Run x2 c aus e s abn ormal different iatio n of cho ndroc yte s re sulti ng in sk elet al dw arfis m. The major hi ston e rela ted to t his p ath of e v ent s is hi sto ne H3. H3 normally sit s on th e Runx2 pr omot er, inhibiti ng the Run x2 ge ne activ ity an d su bs equ ent prod ucti on of Runx 2 protei n in prolif eratin g cell s. With H DA C4 mi ss ing in th e kno ckout (V ega, Mat sud a et al. 2004), H3 i s hyper- ac etyl ated, le av es t he Runx2 pr omoter and th e Runx 2 gen e is a ctiv ate d. With H DA C4 intac t, hypo-a cet ylat ed H3 intera ct s with D NA an d HS more, ke ep ing it on the Run x2 prom oter, inhibiti ng R unx2 pro duct ion an d prom oting co ntinu ed c hondro cyt e proliferat ion a nd redu ced d ifferen tiati on to hyp ertrophi c st ate s. With th e import anc e of perle ca n and n ow H3 in ret ainin g chondr ocyt e prolif eratio n, perle can mi ght aga in be in a tern ary compl ex of neg ativ e DN A, pos itive h is tone H3 and th e neg ativ e HS, a compl ex th at inhi bit s Run x2 ge ne a ct ivity an d kee ps chon drocy te s proliferat ing un til ce ll si gnal s trigg er hypertro phy.

Outline The remai nder of thi s di s serta tion wi ll prese nt the fin ding s of my res earc h proje ct of ch arac teri zing t he bindi ng of perl ec an to known and nov el bin ding p artner s u sin g the growt h plat e cart ilag e from fetal bo vine rib s a s our mod el s y stem. My first sp ecifi c aim was t o stu dy th e bindi ng of FGF-2 to g rowth pla te perl ec an an d to determi ne wh at porti on s of perle ca n ar e involv ed in FGF- 2 bindi ng and whi ch port ion s are bl ocki ng the d e livery of thi s grow th fac tor to its re cept or as h ad be en pre viou sly d is cover ed. The se findi ng s are

34 summ ariz ed in Pap er I. The sp ecifi c ai m for Paper II wa s to ob serv e wheth er perl ec an cou ld bind t o FGF-18 . In that paper, I sh ow th at perle can c an bi nd to FGF-18 and I ch a racteri ze th e kin etic s an d spe cific ity of the ir bindi ng. For Paper III, my specifi c aim w as t o find nove l bindi ng part ner s of perle ca n that are e ndog eno us to t he growth pl ate. Hi sto ne H3 i s pre se nted in Paper III a s a third k ey deve lopm enta l protei n to whi ch grow th plate p erlec an bi nds. A brief di scu s sion of th e maj or finding s of my di ss ertati on rese arch fo llow s Pap er s I-III. Included after thi s dis cu ss ion i s an Appe ndix c onta ining a revi ew pa per re garding t he role of p erle can in regul ating o cul ar functi on.

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CHAPT ER TWO

PAPERS

Paper I

Hepar an and Chond roitin Sulfat e on Growth Plate Pe rle can Mediat e Bindin g and Del ive ry of FGF-2 to FGF Receptor s

Simone M-L Smith * , Leigh A. W est ‡ , P rasa nthi G ovindr aj ‡ , Xiuqi n Zhang ¶ , D avid M. Ornitz ¶ and Joh n R. Has s ell * ‡

* Depart ment of Mol ecul ar M edic ine, C olleg e of Me dici ne, Univer sit y of South Flori da, Tamp a, FL. ‡ Cent er for Re se arch i n Skel etal D ev el opment and Pe diatri c Orthop aedi c s, Shriner s Ho spit als for Childr en - Tamp a, FL. ¶ Dep artme nt of Mo lec ular B iolo gy and Pharmac olog y, Wa shing ton Univer sit y Scho ol of Me dici ne, St. Louis, M O.

Abbrev iati ons u s ed: C AF, cati onic fil t ration; CS, chon droitin sulfa te; D DSH, dy ss egm enta l dy spla si a , Silverman-H and mak er type ; DME M, Dulbe cc o’s mo dified Ea gle m e dium; D MM B, dimeth ylmet hyl ene bl ue; FGF, fibro bla st growt h fact or; FGFR, fibrobla st gro wth fa ctor rec eptor; GAG , glyco samin ogly can ; HS, hepar an s ulfat e; HU AEC, hum an umbil ical art erial endot heli al c ell; 1 2 5 I, Iodine-125; IP, immuno prec ipit ati on; SJS, Sch wart z-J amp el syndr ome.

Publi shed i n Matri x Biolog y 200 7 Apr; 26(3):175-8 4. Epub 2006, Nov 6.

48 Abstr act

Fibrobla st gro wth fa ctor (FGF)-2 reg ul ate s cho ndroc yte proliferat ion in th e growt h plat e. Hep aran s ulfat e (HS) proteog lyc an s bind FGF- 2. Perlec an, a hepara n su lfate pr oteo glyc an (HSPG) in the d eve lopin g growt h plat e , however, c ontai ns b oth HS and ch ondroi tin s ulfat e (CS) ch ain s. The bindi ng of FGF-2 to perle can i sol ate d from the gro wth pla t e wa s ev alua ted u sin g cati onic filtration ( CAF) an d immun opre cipit ati on (IP) as say s. FGF-2 bou nd to perle can i n both th e CAF and IP a ss ays pri marily v ia th e HS chai ns on p erle can. A ma ximum of 12 3 molec ule s of FGF-2 w as calc ulat ed to bi nd per mol ec ule of per l eca n. When d ige st ed with chondr oitin as e AB C to rem ove it s CS c hain s, perle ca n augm ent ed bindin g of FGF-2 to the F GFR-1 and F GFR-3 re cept ors and al so incre as ed FGF-2 stimu lati on of [ 3 H]-th ymidin e incor porati on in BaF3 cell s ex pre ssi ng th es e FGF rec ep tors. Thes e dat a s how th at growth pl ate p erle can bi nd s to FGF-2 by its HS chai ns b ut ca n only deliv er FGF-2 to FGF re cep tors when i ts CS c hain s ar e remov ed.

Keyw ords : growth p lat e, perle can, FG F, FGFR, chon drocyt e, chondr oitin sulfa te, hep aran sulf ate.

49 Introduct ion

Perlec an w as ori gina lly id entifi ed a s a hepara n su lfate proteog lyc an (HSPG) in t umor lin es a n d was sho wn to b e pre sent i n all ba sem ent m embra ne s (Ha s sell, R ob ey et al. 19 80; Ioz zo an d Wight 19 82). It is no w know n not onl y to be pre sent i n the extra cell ular matr ix of carti lag e and i n the growth p lat e, but al so to be one of th e prin cipa l medi ator s of lo ng bone gr owth (Sun dar Raj, Fite et al. 19 95; H andl er, Yurch enc o e t al. 1997; Fren ch, Smith et al. 1999). Perle can n ull mic e hav e red uced chon drocyt e proliferat ion a nd matri x depo sit ion in the dev elo ping gro wth pl ate, which r esu lts i n fetal d warfi sm (Arik a wa-Hir as awa, W ata nab e et al. 1999; C os tell, Gu st afs son et al. 199 9). Mutati ons i n the hu man perle can g ene cau se t wo ch ondrod ys tro phie s: dy ss egm enta l dysp la sia, Silv erma n Han dmak er typ e (DDSH) an d Sch wartz- Ja mpel syndr ome (S JS) (Nicol e, Dav oine et al. 2000; Arik awa- Hira sa wa, Wilco x et al. 20 01; Arik aw a-Hira sa wa, Le et al. 2002). DDSH rese mble s ma ny fea ture s of the p erle ca n null mou se a nd re sult s from an ab se nce of fun ctio nal p erle can. SJ S is le ss se vere th an D DSH bec au se eit her a trun cat ed form of perl eca n is ma de or red uce d level s of full-l engt h perle ca n are ma de . These ob serv atio ns in dic ate that perl ec an pl ay s an e s senti al rol e in endoc hondr al o ssifi cat ion. The role of perl ec an in ba s emen t mem brane s i s sum mariz ed in a recen t revie w (Ioz zo 2005). Fibrobla st growt h fact ors (FGF s) are a family of prot ein s that reg ulat e cel l prolifer ation and diff erenti ation d uring e mbryon ic deve lopm ent by bi nding t o FGF rec ept ors (FGF Rs) (Orni tz an d Itoh 2001). FGF-2 (Nug ent, Nug ent e t al. 2 000) is pr es ent in th e deve lopin g growth p lat e (Hill an d Log an 1992 ; Luan, Praul e t al. 1996) wh ere it c an regu lat e cho ndroc yt e prolifer ation a nd th ereby

50 affect lo ng bon e growt h (Trippel, Wro blew ski e t al. 1993 ; Nag ai, Tsukud a et a l. 1995). Whil e the F GF-2 knocko ut mou se h as n o sev ere d efec t in long b one gro wth (Do no, Texido et al. 1998; Orteg a, Ittmann e t al. 1998; Zh ou, Sutl iff et al. 1998), sh owin g only a mild, adul t-on set o ste ope nia (d ecre as ed bon e-miner al de nsi ty an d bone form ation) ( Mont ero, Oka da et a l . 2000), the FGF-2 tran sg eni c mous e, wher e FGF-2 i s over-e xpre s sed , has s ever ely short ene d limb s resul ting from c hondro cyt e hyp erpla si a, incre as ed extr ac ellul ar matrix de po sitio n and d ecre a sed diff er entia tion of hy pertrop hic chondr ocyt e s (Coffin, Florki ewi cz e t a l. 1995). The ab sen ce of lon g bone d efec ts in t he FGF-2 k nock out c o uld be du e to com pen sa tion for FGF-2 by other F GFs in t he grow th plate. Perlec an ma y play a role in re gula ting the di stribut ion or avail abili ty of FGF s in the gr owth pl at e (Arika wa-Hir as aw a, Wata nab e et al. 19 99; Ol se n 1999). FG F-2 bind s to hep arin an d hepar an s ulfat e in a ma nner oft en de pe ndent u pon th e numb er and seq uen ce of th e su lfate est er s in the gl yco sami nogly ca n cha in (Ornitz a nd Leder 1 992; Po well, Y ate s et al. 2004). FGF-2 bin ds t o perle can m ade b y an en doth elia l cell li ne (Knox, Merry et al. 2002) and to per lec an ma de by gro wth pl ate chondr ocyt e s in cult ure (Govindr aj, We st et al. 2006) a nd the bindin g is to th e hep aran sulfa te ch ain s. Endoth eli al cel l perle ca n conta in s only h epar an sulfa te ch ain s (Kno x, Merry et al. 200 2) but perle ca n iso lat ed from the growt h plat e al so c ontai ns chon dro itin sulf ate chai ns i n addit ion to the he para n sulf ate chai ns (G ovindr aj, We st et al. 20 02). The chondr oitin sulfa te ch ain s on gro wth p late p erlec an h ave re cen tly been sho wn to bin d to coll ag en an d ac celer ate coll agen fibr il formation (K vis t, Joh ns on et al. 20 06). FGFs bi nd to bot h low an d high affi nit y recep tors ( Ma sc arell i and C ourtoi s 199 3). We s how ed tha t p erlec an i s the lo w affinit y

51 recep tor for FGF-2 in th e dev elopi ng g rowth pla te an d that F GF-2 bound to p erle can w a s not de liver ed to the high affi nity re cept ors o n growth pl ate c hondr ocyt e s (Govin draj, West et al. 200 6). There are four high affini ty rec eptor s for FGF-2 (FGFR-1, 2, 3, and 4) an d variou s s plic e vari ant s of ea ch (FGF R- 1b, FGFR-2 c, for examp le). FGFR-1 a nd FGF R-3 are th e FGF Rs ex pres se d in the d eve lopin g growth pl ate (W ang, Gre en et al. 2001) . FGFR-1 is u pregul ate d in the growt h plat e a s cho ndroc yte s hyp er trophy an d FGFR- 3 is expre s sed in pr olifer ating chon drocyt e s. A mis se ns e mut ation in FGFR-3 i s kno wn to c au se a chon dropl a sia, th e mos t comm on form of human ske let al dw arfism (Shi ang, Tho mpso n et al. 199 4). A rare mutatio n in FGF R-1 al so c au se s os teo g lophon ic dy spl as ia, a s ever e dis ea se c hara cteri zed b y dwarfi sm, pre mature fu sio n of crani al bone s an d bone l e sion s (Whit e, Ca bral et al. 2005). Ot her hum an ske let al dy spla si as h av e bee n link ed to FGFR-1 (M uenk e an d Sche ll 1995). FGFR- 1 and FGF R-3 th us h ave disti nct e xpre s sion do main s in the grow th pla te an d perform un ique function s. In this st udy, we u s ed a c ation ic filtra tion a ss ay (For sten, Wang et al. 2000) and an i mmuno prec i pitatio n a ss ay (Whi telo ck, Murdo ch et al. 1996) to te st th e bin din g of FGF-2 to purifie d perle can from th e dev elo ping gro wth p late. W e al so us ed a n immunopr eci pitat ion a s say w ith rec om binant r ece ptor s FGFR- 1c an d FGFR-3 c and a pr olifer ation as sa y wit h BaF3 cell s ex pre ssi ng th es e recep tors t o mea sur e the a bility of gro wth plat e perl ec an to de liver FGF-2 to FGF R.

52 Expe rimenta l Proc edur es

Chemic als Rec ombin ant h uman F GF-2 wa s from P eproTec h (Ro cky Hi ll, NJ, U. S. A. ). Protein G Seph aros e 4 Fa st F low and Hep arin Sep haro se 6 Fast Fl ow wer e from Amer sh am Bio s ci enc es (Pi sc ata way, N J, U. S. A. ). Chondroit ina se A B C and pro t ea se-fre e cho ndroiti na se A BC (both EC 4. 2. 2. 4), chon droitin a se A CII (EC 4.2. 2.5), heparitin as e I (EC 4. 2. 2. 8) and hep aritin a se II (no EC numb er) were from As soc iat es of Cap e Co d (East Fa lmout h, MA, U.S.A. ). Lipofect amin e plu s wa s from Invitr oge n. DMEM w a s from Gibc o. 1 2 5 Sodium [ I ]iodide ( carrier fre e, 17. 4 Ci/mg) an d [methy l- 3 H]thymidi ne (6. 7 Ci /mmol) w ere from PerkinElmer (B ost on, M A, U. S. A. ). Chemic als were from Sig ma ( St. Louis, MO, U.S.A.) unle ss o ther wis e indi cat ed.

Perle can Growth pl ate s from third trim e ster fet a l bovine rib s w ere di s sec ted and the pr otei ns e xtra cted with 4 M gu anidin e hydr ochl oride. The perle can in t he ex tract wa s purifie d us ing C sCl d en sity gr adie nt centrifu gati on follo wed b y ion ex ch an ge and siz e ex clu sion chroma togra phy a s pre viou sly d es crib e d (Govindr aj, We st et al. 2002). The purifie d perl eca n wa s dia l yzed o verni ght at 4º C ag ain st disti lled w ater to r emov e the gu anid in e and al low th e nati ve struc ture of th e core pr otein t o reform (Ledbett er, Fish er et al. 1987) a s s een in We ster n blot s of the f inal prod uct. The glyco sa minog lyc an co nten t of the purif ied perl ec an wa s d etermi ned by the dim ethy lmeth yle ne blu e (DM M B) dye bi nding as sa y us ing chondr oitin sulfa te C as a sta ndar d (Fa rndale, B uttl e et al. 198 6).

53 Soluble FGF Re cepto r Con stru cts enco ding th e s olubl e FGF R 1 and FGF R3 rec ept ors w ere obtain ed from Dr. D. Ornit z (Ornitz, Y ayon et al. 1992). Eac h con struct wa s com pris ed of the extra ce llular regi on of the r esp ect ive murine re cept or fus ed to pl ace ntal alka line pho sp hat as e (AP) and clon ed into a n expr es sio n ve ctor. Co s7 cell s were gr own in D ME M supp lem ente d with 1 0% FBS. The FGF Rec ept or-Alk aline Phosp hat as e (FR AP) pla smid w a s tran s fecte d into c ell s us ing lipofe ctami ne plu s. The am ount s of rec ombina nt rec eptor pro duc t se crete d into th e med ia wer e det ermin e d by mea surin g the a lkal ine phos pha ta se a ctivi ty in the m edi a.

Prepar ation of Radi olab elled FGFs 1 2 5 FGFs w ere radi ola bell ed u sing sodi um [ I ]-Iodide activ ate d in IODO-GEN- coat ed tu be s (Pierc e Bio te chnol ogy, Ro ckford, IL, U. S. A. ) using th e man ufac turer’ s su ppl ied proto col (C hiz zonit e 1 2 5 indire ct meth od). Briefly, 0.5 m Ci of sodi um [ I]-Iodide w as adde d to the IO DO-GEN t ube c onta inin g 100 µl of iodin atio n buffer (0.025 M Tris- HCl, 0. 4 M Na Cl, pH 7. 4). After 6 min at room temper ature, th e ac tivat ed io dine solu t ion wa s tran sferr ed to a 1.5 ml polypro pyle ne tub e co ntain ing 5 µg of FGF-2 in 100 µl iodina tion buff er. After rea cting for 9 min at room tem perat ure, the reacti on w as t ermina ted by t he ad ditio n of 50 µl of 10 mg/ml L- tyrosi ne. The s pe cific a ctiv ity of the r adiol abel led FGF- 2 wa s determi ned b y as su ming a ll wa s iodi na ted and sub jec ting 1% of th e iodina ted pro duct t o SDS-PAGE, de ter mining th e loc ation of t he FGF-2 by aut oradi ograp hy, ex cis ing th e band and co untin g the radioa ctivi ty in a Mod el 1191 g amm a c ounter (TM A nal ytic, Elk Grove Vi llag e, IL, U. S. A. ). The remaining s ampl e volum e wa s adju ste d to 0. 4 ml with colum n buffer (iodinat ion buff er cont aini ng

54 1 2 5 0. 1% [w/v] BSA), and u nin corpora ted Iodine remo ved o n PD-10 column s (Am ers ham B io sci enc es) e quil ibrate d in colu mn buffer. Aliquo ts of fra ction s w ere co unte d to determi ne th e elut ion po siti on 1 2 5 of Iodine inc orpora ted int o FGF-2. Fractio ns c ont ainin g radiol abell ed FGF- 2 were p oole d and i ncub ated with H epari n Sephar os e 6 Fa st Flow b ead s (Am ers ha m Bio sci enc es, pre wa sh ed with 4 M G uani din e HCl t hen P BS) for 1 hour with ge ntle ro ckin g. The bea ds w ere th en w ash ed wi th PBS and the b ound F GF-2 rele a sed with elu tion buff er (2 M Na Cl c onta ini ng 0.025 M Tri s HC l, pH 7. 4). The eluat e wa s dia lyz ed ag ain st PBS, and th e inc orporat ion of 1 2 5 Iodin e into FGF-2 w a s verifi ed by c ountin g in a gam ma co unter and by SDS-P AGE and autor adio graph y to confirm th e mole cul ar mas s of the i odin ated gr owth fa ctor. The dial ys ed el uat e wa s the n o 1 2 5 store d in ali quot s at –8 0 C un til us e. [ I]-FGF-2 prepare d usi ng this pro ced ure ha d com para ble bi nding chara cteri sti cs t o 1 2 5 commer cial ly prep ared [ I]-FGF-2 (r ecom bina nt hum an, Bolt on- Hunter l abel led; P erkinElm er) whe n te ste d for perle can bi ndin g in the ca tioni c filtrati on a s say d es crib ed below (d ata n ot sh own).

Cationi c filtration a s say Perlec an-FGF- 2 bindi ng wa s d etermin e d usin g a ca tioni c filtrati on (CAF) a ss ay (For sten, W ang e t al. 200 0). Binding a s sa ys w ere performed i n a buffer c on sis ting of 0. 0 5 M Tris-H Cl, 0.15 M N aC l, 1 2 5 2 mg/ml prote a se-fre e BSA, p H 8.0. [ I]-FGF2 was i ncu bat ed in the ab se nc e and pr es enc e of perl eca n a nd comp etitor li gand s in o solut ion at 2 5 C for 1 h in a fin al inc u bation v olum e of 0. 2 ml. 1 2 5 Contro l perle ca n and p erle can bo und t o [ I]-FGF-2, were cap tured by vac uum filtra tion a cro ss a cati onic membra ne (Zeta-Pr obe; Bio- Rad Lab oratori e s, Hercu le s, CA, U.S. A. ) place d into a 9 6-wel l dot-

55 blot app arat us ( Minifol d I; Schl eich er and Sch uell, K een e, NH, 1 2 5 U. S. A. ). Unbound [ I]-FGF-2 wa s re moved b y rinsi ng the well s three tim es with 0. 2 ml in cub ation b uf fer. The filters w ere drie d and dot s corr es pondi ng to e ach well w ere cut o ut, plac ed int o tube s and the r adio acti vity me a sured i n a ga mma cou nter. As s ays were 1 2 5 performed i n a minim um of dupli cat e s. Binding of [ I]-FGF-2 to the mem bran e in the a bs enc e of perl ec an wa s s ubtra cte d to determi ne spe cific ally b ound gro wth fa ctor.

Antis era Polycl onal anti serum t o perle ca n (GPP 76) and pol ycl onal anti serum to aggre ca n (RB 012) w ere obt aine d by purifying p erle can and aggre can from fe tal bo vine rib gr owth plate s (Go vindr aj, We st et a l. 2002) an d empl oying a comm erci al s er vice (Invi troge n; C arls bad, CA, U.S. A. ) to rais e ant ibodi e s to the s e proteo glyc an s in rab bit s. Anti-c alf inte sti nal a lkali ne ph os phat a se a ntibo dy wa s from Sigm a (St. Louis, MO, USA).

Dot Blot The cap acit y of Zeta-Pro be mem bran es to bind perl ec an in the CAF as sa y wa s det ermin ed by filt ering sam ple s cont aini ng differ ent amount s of per lec an thro ugh th e memb rane an d wa shi ng the filt ers as d es cribe d abo ve. After dryi ng, the filters w ere bl ock ed for 1 h in Dulbe cc o’s P BS (Me diat ech, H erndo n, VA, U.S. A. ) contai ning 0. 2% (w/v) I-Bloc k (Appli ed Bi os yst em s, Fost er City, C A, U. S. A. ). The primary an tis erum (GPP7 6) wa s ad ded at a final con centr ation of 1:400, an d the blo t incu bat ed for 18 h at 4°C. The p erlec an sign al was d et ect ed us ing a 1: 5000 d ilutio n o f a horser adi sh per oxid as e- coupl ed s ec ondar y anti body and an E C L kit (Amersh am Bio sci enc e s). Image s were re cord ed o n x-ray film (Bio M ax XA R;

56 Koda k, Roc he ster, NY, U. S.A. ) and p i xel den sit ie s of dot s in the linear ra nge of film e xpo sur e were m ea sure d usi ng Qu antit y One softw are (B io-R ad Labor atori es).

Immunopr ecip itation a ss ay FGF bindin g to perl ec an wa s al so eval uate d usi ng an immunopr eci pitat ion (IP) as s ay for per leca n sim ilar to th at previou sl y de scrib ed (Wh itelo ck, M urd och et a l. 1996; K nox, M erry et al. 2002), ex ce pt that at the c ompl et ion of the immunopr eci pitat ion, the b ound [ 1 2 5 I]-FGF-2 wa s fract iona ted in to that el uted b y 2 M N aCl a nd th at remai ning bou nd to th e bea ds. This co nce ntrati on of s odium c hlorid e was se lec ted b ec aus e it ha s been sho wn to rel ea se FGF- 2 boun d to hepari n and to t he lo w affinity FGF re ce ptor pre se nt on c ell s and in ti ss ue s (Chi ntal a, Mill er et al. 199 4; Chi ntal a, Mill er et al. 1995). Unle s s oth erwi se 1 2 5 noted, 12 8 ng of perl eca n wa s in cuba te d with 10 5 cp m of [ I]-FGF- 2 in a final vo lume of 1 ml of IP buffe r (0. 02 M Tris, 0. 15 M o sodi um chl oride, 1% (v/v) Triton X-1 0 0, pH 7.4) for 1 h at 25 C. Five micro liter s of eit her norm al rabbi t (non-immun e) or immun e serum wa s then add ed an d the sampl e s were in cub ated for 3 h a t o 4 C. Twenty-fiv e micro liter s of Protei n G-conj ugat ed Se pharo s e 4 Fast Fl ow be ad s, wa she d twic e in 1 ml IP buffer by centrif ugat ion o for 5 min at 2500 g at 4 C, wer e add ed to each t ube a nd the t ube s o tumble d overn ight at 4 C. The be ad s w ere the n wa sh ed thre e time s as a bov e with 1 ml IP buffer a nd the b ound mat erial elut ed by incub atio n with 0.5 ml of 2 M sod ium chlorid e (in IP buffer wit hout o Triton X-100) for 30 min at 25 C, foll owed b y cen trifuga tion a s o abov e at 25 C. The radio activ itie s in t he su pern atan ts (e lute d

57 count s) an d pell et s (remai ning c ount s) were me a sured i n a gamm a count er.

Glyco sida se t reatme nt of perl ecan For CAF an d IP as sa ys, per lec an (128 ng of DM MB r eac tivity) w a s either sha m dige st ed, or dige st ed s epar ately w ith eit her prot ea se- free cho ndroiti na se A B C (10 mU) to re move ch ondro itin s ulfat e glyco sa minog lyc an (GA G), a mixture o f hepariti na se s I and II (5 mU eac h) to remov e HS-GA G, or with prot ea se-fre e cho ndroiti na se A BC and he pariti na se s to remo ve bo th CS- a nd HS-GA Gs, in 0. 1 ml dige stio n buffer (20 m M Tris-H C1, 50 mM s odium a cet at e, 5 mM calc ium ch lorid e and 0. 2 mg/ m1 BS A, pH 7.4) for 3 h at 37° C. Compl etio n of glyc osi da se di ge stion s was confirm ed by th e lo ss of DM MB re act ivity of shar k cartil ag e ch ondroiti n sulf ate C or bovin e kidne y HS (5 µg ea ch) in dig es tion s s e t up in paral lel wi th appropri ate enzy me s. Bindi ng of sh am and glyc os ida se-tr eat ed perle can to t he Zet a-Probe m embra ne was confirm ed by do t blot a s des crib ed abo ve. For ce ll prolif eratio n as say s, perl ec an (100 µg by DM MB re act ivity) or 10 0 µg of chon dr oitin s ulfat e C w ere dig es ted with 50 mU of pro tea s e-free c hondro iti nas e A BC in 1. 5 ml of 25 m M sodi um ac etat e, pH 7. 0, for 1 h at 37° C.

FGF Recepto r Bindi ng A ss ay The bindin g a ss ay mixt ure co nsi st ed of conditi one d medi a from Co s 7 cell s tha t were tr an sfec ted wi th co ns truct s enc odin g FGF Rec ept or-Alk aline p ho sph ata se or F RA P (0. 4 OD unit s of phos pha ta se a ctivi ty for FRAP- 1c an d FRAP-3 c), [ 1 2 5 I]-FGF-2 and perle can. All re act ion s wer e inc ubat ed at room temp eratur e. Undig es ted p erle can, ch ondroi tina s e-di ges ted p erle can or h epari n was add ed in co nc entrat ion s rangi ng from 0 to 2.5 µg GAG s / tub e

58 (0-10 µg GAG s / ml). Anti- calf int es ti nal alk alin e pho sph ata s e antibo dy wa s a dde d to immun opre cipit ate th e FRAP and 20 µl of Protein G sep haro se w a s us ed to c aptur e the an tibod y. The be ads were w as hed t wic e with ic e col d PBS a nd the tub e s were coun ted directl y in a ga mma c ount er to mea sur e the am ount of iod inat ed FGF2 ca pture d on the b ead s. For ea ch reacti on co nditi on, a con trol was d one with id enti cal re age nts and p re-immun e ser um in ste ad of the ant ibod y. The res ulting v alu es for the pre-imm une control s w ere subtr act ed from the exp erimen tal v alue s to giv e the spe cific v alu es sho wn in our re sult s.

Cell pr olife ration a s say The abilit y of perl eca n to medi ate t he deliv ery of FGF-2 to FGF rece ptor s wa s det ermin ed u sing B aF3 cel ls as pr eviou sl y des crib ed (Ornit z, Yayo n et al. 19 92). In brief, BaF3 c ell s transf ect ed wit h pla smid s c onta ining t he FGFR- 1c an d FGF R-3c con struct s w ere w as hed wi th RP MI 164 0 medium l acki ng IL-3 and plate d in 96-w ell micr otiter pl ate s at 2 x10 4 cel ls/ well. FGF-2 a t 200 pM and ei ther h epari n, perle can, p erlec an dig e sted w ith protea s e-free c hondro itin as e AB C or c hondroit in su lfat e dige st ed with prot ea se-fre e cho ndroiti na se A B C were ad ded a t 0-5 µg/ml, all in 200 µl RP MI-164 0 medi um per w ell. One micro curie of [ 3 H]- thymidi ne in 50 µl of RPMI-1 640 w as adde d 36 h lat er and, aft er 6 h of culture, c ell s wer e coll ect ed wit h a PHD ce ll harv es ter (Cambri dge Te chno logi es, Inc. , W atert own, MA, U.S.A. ). Incorpor ated [ 3 H]-th ymidi ne w as d eter mined by li quid sci ntill ation counti ng.

59 Res ults

Binding of FGF-2 to Perl ecan in the CAF As say Prelimin ary exp erime nt s were p erform ed to optim ize t he am ount of perle can u se d in the CAF a s say. We fi rst te ste d the bi nding cap acit y of the c ation ic mem bran e for perle can b y filterin g differe nt amount s of per lec an a nd me asur ing th e amount bo und to th e filter with the anti seru m to perl eca n. The re sult s indi cat ed th at the maximum amou nt of perle ca n that c oul d be retai ned o n the filt er was ~8 ng (Da ta not sho wn). Quant iti es of 1. 6 ng to 8 n g of perle can w ere u se d in incu bati on s cont aining p erle can, a nd incub atio ns w ere al low ed to pro cee d for one hour in all su bs equ ent bindin g and c ompe tition as s ays. The b i nding of [ 1 2 5 I]-FGF-2 to 2 ng perle ca n wa s det ermin ed at c onc ent ration s rang ing from 0. 086 t o 44 nM [ 1 2 5 I]-FGF-2 (Figur e 5). After s ubtrac ting b ack ground bindin g of FGF-2 to the Ze ta Prob e me mbrane (Fig ure 5, “no perle can ”) we fou nd appr oxim atel y 1. 6 nanogr am of FGF-2 bou nd per nan omolar F GF-2 add ed in th e ran ge of 0. 172 to 22 n M FGF-2 adde d until sat uratio n wa s rea ch ed at 2 2 nM.

HS particip ate s in perl eca n-FGF-2 binding Perlec an w as either l eft undi ge ste d, or dige sted sep arat ely wi th either pro tea s e-free c hondro itin as e AB C or a mixtur e of hepari tina s es I an d II. Undige st ed or g lyco sid as e-tre ated p erle can was t hen in cub ate d with 4 ng of [ 1 2 5 I]-FGF-2, and s pe cific bi nding determi ned. Bi nding of [ 1 2 5 I]-FGF-2 t o perle can w a s marke dly reduc ed by h eparit ina se d ige stio n, indi catin g a requ ireme nt for hepar an s ulfat e ch ain s in perl ec an-FGF -2 binding (Fig ure 6). Chon droitin a se dig es tion in cre as ed [ 1 2 5 I]-FGF-2 binding t o perle ca n

60 sligh tly but n ot si gnific antl y. Equival ent bind ing of und ige st ed and glyco sid a se-tre ate d perl eca n to the Zet a-Probe m embran e wa s confirme d by immu noblo tting (d ata n ot show n). The ability of fre e chondr oitin sulfa te or he para n sulf ate chai ns to comp ete for th e bindin g of perle can t o FGF-2 wa s al so eval uat ed. Perlec an w as adde d to tub es c ont ainin g 0 – 200 ng o f chondroi tin s ulfat e C or bovin e kidn ey HS, follo wed by t he ad d ition of 1 ng of [ 1 2 5 I]-FGF-2. After one h our of inc ubati on th e sa mpl es w ere filt ered a nd sp ec ific bindin g det ermine d. Hepar an s ulfat e reduc ed [ 1 2 5 I]-FGF-2 bindi ng to perle can b y 90% but di d not ab oli sh binding e ven at 1µg/ml (Figure 7). In contra st, ch ondroi tin s u lfate did n ot redu ce bi ndin g. This co nfirms th e re sult s obt ain ed by dige stin g the p erlec an wi th chondr oitin as e AB C or he pariti na se s a nd mea surin g FGF-2 bin ding (Figure 6).

Binding of FGF-2 to perl ec an in the IP as say The line arity of th e IP as say wa s first determi ned b y comb ining 1 2 5 perle can w ith [ I]-FGF-2 (1µg/10 6 c p m), and immuno preci pita ting aliquo ts of thi s mix ture c onta ining 2 5- 500 ng of perl ec an u sing a nti- 1 2 5 perle can serum and prot ein G b ead s. The amou nt of [ I]-FGF-2 elute d from the b ead s wit h 2 M s odium chlorid e and th e amo unt remaini ng on th e be ad s wa s the n mea s ured. Approxi mat ely 70% of 1 2 5 the [ I]-FGF-2 bound t o bea ds i n the pres enc e of the anti serum t o perle can w a s elut ed by 2 M so dium ch l oride (Figur es 8 A and B). Approxi mat ely two fol d more [ 1 2 5 I]-FG F-2 was r ele as ed by 2 M sodi um chl oride from b ead s in cub ated with thi s ant is erum to perle can th an from be ad s inc ubat ed wi th an ant is erum to ag gre can or bead s in cub ated w ithou t s erum throu gh out the ra nge of p erlec an test ed (Figur e 8A). This ind ic ate s that this IP a ss ay i s line ar for up to 500 ng of perl ec an.

61 We the n us ed th e IP as sa y to det ermin e the role of t he he para n 1 2 5 sulfa te or ch ondroit in s ulfat e ch ain s o n perle can in b indin g [ I]- FGF-2, whic h wa s su bs equ entl y rele as ed by 2 M sodi um chl oride and me as ured (Fig ure 9). Chon droitin as e dige sti on en han ced FG F-2 bindin g by 20%, but be ca us e thi s incr e as e wa s s een w ith bot h the non-immu ne s erum a nd the p erle can an tiser um, the bin ding i s not signif ica nt. Hep aritin as e dig es tion, ho wever, red uc ed the b indin g of 1 2 5 [ I]-FGF-2 to perlec an by 8 5%.

Binding of FGF-2 to FGF Rec eptor s: Cell-fr ee A s say Previou s st udi es h ave show n that th e formation of a trimol ecu lar compl ex co ns ist ing of h epari n/he para n sulfat e, FGF R and F GF co uld be requ ired for c ell re ce ptor-me diat e d sig nalin g to o ccur (Or nitz, Yayo n et al. 1 992; Ch ella iah, Yuan e t al. 19 99). Sin ce F GF-2 w a s sho wn to bi nd p erlec an in both t he c a tionic fil tratio n a s say and th e immunopr eci pitat ion as s ay in thi s stu dy, we first te ste d th e a bilit y of perl eca n to enh anc e FG F-2 bi ndi ng to F GFR-1 c and F GFR-3 c usin g the immun opre cipit atio n a ss ay. The sp ecif ica lly bo und F GF-2 is sho wn a s a fun ctio n of th e G AG co ncen tratio n ad ded (Figure 10). For both r ec eptor s, ad ditio n of p erle ca n ca us ed a s tea dy d ecre a se i n the amoun t of FGF-2 that bo und th e FGF rec eptor. I n c ontra st, additi on of ch ondroit ina se- dige st ed p erlec an in crea s ed the amou nt of FGF-2 that bo und to ea ch of th e re cept ors. Th e addit ion of hepari n in cre as ed t he bindi ng of FGF -2 to F GFR-1 c b ut d ecr ea se d the bind ing of FGF-2 to F GFR-3 c.

Binding of FGF-2 to FGF Rec eptor s: Effect on c ell pr olife ration We al so te st ed th e abili ty of perl eca n cont ainin g bound F GF-2 to form a compl ex wit h FGFR and e nhan c e the mito geni c a ctivit y of FGF-2 on B aF3 c ell s expr es sin g FGFR -1c or FGFR-3 c. Perlec an di d

62 not stim ulat e inc orpora tion of [ 3 H]-thy midine in BaF 3 cell s expre s sing FG FR-3 c (Figure 1 1, left p anel) bu t did prod uce a sli ght stimul atio n of [ 3 H]-thymid ine in corpor ation a t the hig he st conc entr ation of p erle can in BaF3 cell s expr es si ng FGF R-1c (Figur e 11, right pan el). In contra st, perl ec an dige ste d with c hondr oitin as e AB C to remov e the chon droitin sulf ate chain s s timul ated [ 3 H]- thymidi ne in corpor ation in b oth B aF3 cell lin e s. The chondr oitin as e AB C-dig est ed ch ondroi tin s ulfat e con t rol did not s timul ate incorpor atio n in eith er cel l line.

Dis cus sio n

The res ults of t he se studi es u si ng both a catio nic filtr ation (CAF) a ss ay an d an imm unopr ecip itati on (IP) as say sho w that perle can p urified from th e dev elo ping growth pl ate bi nd s to FGF-2 and thi s indi cat e s that n o other e xtra ce llular matri x com pone nt s are need ed for thi s bind ing to o ccur. Thi s bindin g is prim arily to th e hepar an s ulfat e ch ain s on perl ec an. R emov al of the h epar an s ulfat e chai ns by h ep aritin as e dig esti on mark e dly decr ea se d FGF-2 bin ding and com peti tion studi e s with h epar an s ulfate re duc ed bin ding by 90%. These d ata in dic ate th at the h ep aran s ulfat e ch ain s on perle can ar e e ss enti al for maxi mum FG F-2 binding. The amou nt of FGF-2 tha t coul d bind t o perle can in t he C AF as sa y wa s dep end ent on t he co nc entrat ion of FGF-2 u sed (Fig ure 5). We foun d that a t a con ce ntrati on of 22 nM FGF-2, (at whi ch bi nding to perle can wa s s aturat ed) 35 ng of F G F-2 could bi nd to 2 ng of perle can. The am ount of p erlec an u se d in the se a s say s i s ba sed o n its gly co sami nogl yca n con tent. B as ed on the ma s s of perl eca n’s

63 core prot ein (No ona n, Fulle et al. 1991 ; Murdo ch, Dodg e et a l. 1992) an d the ma s s an d numb er of the glyco sa minog lyc an ch ain s (Dolan, Hor char et al. 1997 ; Tapan ad e chopo ne, Ha s sel l et al. 199 9; Govindr aj, We st e t al. 2002), perl ec an can b e det ermine d to con tain ~20% glyc os amin ogly can. The 2 ng of glyco sami nogl yca n on perle can (Fig ure 5) wou ld the n corre sp ond to ~10 n g of perle ca n. Estimat ing th e mole cul ar ma ss of p erle can a s ~6 00 kD a and FGF- 2 as ~1 7 kDa, 10 n g of perle ca n bindi ng to 35 ng of FGF-2 wo uld corre spon d to a max imum of 12 3 mole cule s of FGF-2 p er mole cul e of perle can. Thi s woul d sup port th e oft en-propo s ed the ory of perle can a ctin g a s a si nk or res ervoir f or growth fa ctor s in carti lag e and oth er tis su e (Avi ezer, H echt et al. 1994; Ioz zo 19 94; M ongi at, Taylor et al. 20 00; D eguc hi, Okut su e t al. 2002). Previou s work h a s sh own th at he parin can e nhan ce th e dire ct bindin g of FGF-2 to the cell- surfa ce F GF rece ptor (Ornit z, Yay on et al. 1992). We co nfirme d this h ep arin- media ted e nha nce ment of FGF-2 bindi ng to FGF R-1c in t he c ell- free immun opre cipit ation as sa y and to b oth FGF R-1c and FGF R- 3c in the BaF3 pr olifer ation as sa y. While purifi ed p erle can c ould bind FGF-2 in b oth th e CAF and IP a ss ay s, it did not me diat e the F GF-2 stim ulat ion of [ 3 H]- thymidi ne in corpor ation in BaF3 cell s expre s sing FG FR-1 c or FGFR-3 c (Figure 1 1). Howe ver, perl ec an that h ad be en di ge ste d with ch ondroit ina se AB C to remo ve ch ondroiti n sulf ate c hai ns stimul at ed inc orporat ion in thi s c ell li ne. In the FR AP IP as sa y, purified p erle can re duc ed th e bindi ng of FGF-2 to its re ce ptor s, seq ue steri ng the F GF-2 from the FG FR while per lec an th at had b ee n dige ste d with c hondr oitin as e AB C de li vered mor e FGF-2 to bo th FGFR-1 c and FG FR-3 c (Figure 1 0). The data from b oth th e FRAP and the BaF 3 rec eptor a s say s ind ica te that th e chon droitin sulf ate chai ns on p erle can i nhibi t deliv ery of t he boun d FGF-2 to it s

64 recep tors. The s e finding s furth er indi c ate th at perl ec an mad e withou t chon droitin sulf ate c hain s c ou ld deliv er boun d FGF-2 to th e recep tor whil e perl ec an mad e with cho ndroitin sulfa te ch ain s wo uld act onl y a s a si nk for FGF-2. Perlec an ma de by th e EHS tumor a nd b y endot heli al ce lls o nly have h epar an sulfat e ch ain s that are ~7 0kDa a nd ~40 kD a in si ze, resp ecti vel y (Ha ss ell, Ro bey et al. 198 0; Knox, M erry et a l. 2002). Perlec an i sola ted from th e growt h plat e ha s hep aran sulfa te ch ain s of 20 - 22kDa and ch ondroi tin s ulfat e chai ns of 37 - 42k Da (W es t, Govindr aj et a l. 2006) an d of the tot al glyco sa minog lyc an pre se nt, 75% is ch ondroi tin s ulfat e and 2 5% is hepar an s ulfat e (Govi ndraj, We st et al. 20 02). This sugg es ts th at a pproxim atel y half of th e GAG chai ns on gr owth pl ate p erle can would be cho ndroiti n sulf ate and half woul d be h epar an s ulfat e. The seri ne res idu es on t he cor e protein of p erle can th at s erve t o initi at e GAG synt he si s were determi ned b y expr es sin g murine cDN A codin g for the dom ain s of perle can a s re combi nan t produ cts i n eu karyoti c ce lls and th en chara cter izin g the pro duct s. The se stu die s sho we d that th ere ar e three serin es i n doma in I (Co stell, Ma nn et al. 199 7; Dol an, Horc har et al. 1997 ; Sas aki, C ost ell et al. 1998 ) and two serin es in d omai n V (Friedrich, Go hring e t al. 1999 ; Tapan ade chop one, H as sel l et al. 1999) tha t can b e s ub stitut ed wit h a G AG ch ain. The thre e si te s in domain I ar e withi n a sp an of 12 amino acid s, with e ach sit e cap abl e of recei ving ei ther a h epar an sulfat e c hain or a c hondro itin sulfat e chai n (Dola n, Horch ar et al. 199 7). The two gl yco sa minog lyc an site s in do main V are sep arat ed by 35 4 residu e s but an exte nsi ve cys tein e rich re gion b etw een th e two s ites would act to bri ng the m clo ser tog ether. The N-t ermin al sit e i n domain V only re ceiv e s chondr oitin sulfa te ch ain s but th e C-t e rminal s ite c an re ceiv e hepar an s ulfat e. Thes e s equ enc es for G AG att achm ent ar e con s erved

65 in human s (a cc es sio n numb er NP_00 55 20) and bo vine (XP _582 024) perle can. Taki ng all th is to get her, we have compo s ed a s truct ural model of a ty pic al bovi ne gro wth pl ate perlec an mol ecu le ind ica ting the loc atio n and rel ativ e si ze s of th e two type s of GA G ch ain s (Figure 12). Our da ta s how th at th e pres enc e of the c hondro itin sulfa te ch ain s on p erle can i sol ated fro m the growt h plat e prev ent s deliv ery of the b ound FGF- 2 to the re c eptor. We pro po se th at the larger siz e of the se C S cha ins coup led with the ir proximit y to the hepar an s ulfat e ch ain s coul d st eric ally hinder th e tran sfer of the HS-boun d FGF-2 to th e rec eptor (Figur e 12). This s ugg est s th at perle can c ont ainin g chon droiti n sulf at e chai ns could act on ly a s a reser voir for FGF-2 wh ile p erlec an wi t h hepar an s ulfat e only could act to d eliv er FGF-2 to FGF re cept ors; therefore t he ad ditio n of chondr oitin sulfa te to p erlec an wo uld determi ne it s role a s a sink for FGF-2 in the gro wth pl ate ma trix. The degra dati on of perl ec an in the hyp ertroph ic zo ne of th e growth p l ate (W est, Go vindr aj et al. 2006) co uld al so rel ea se t his b ound F G F-2 to promote entry of vas cul ature n ec es s ary to miner ali ze th e matrix in th is z one. FGFR-1 a nd FGF R-3 are dir ect re gul at ors of cho ndro cyte proliferat ion a nd differ enti ation (Orn it z and Led er 1992). The kina se do main s of th es e two re cep tors have v ery s imilar a ctiv itie s in long bon e growt h whe n expr es s ed in gr owth pl ate c hondro cyt es. The differen ce s s een i n our rec eptor studi e s cou ld be du e to th e differe nt con struct s u se d; in the im muno preci pit ation a s say, th e rec eptor s ar e trunca ted by u si ng only t he ex trac ellul ar domai n fus ed to al kalin e phos pha ta se, wh erea s in th e B aF3 a ss a y, the full-len gth re cept or is use d. Als o, con sid ering th e differe ntia l effect s of hep arin on t he two rec eptor s, hep arin i s know n to be cap able of b oth s timul ating and inhi bitin g the bi nding of FG F-2 to FGF recep tors t o affec t cell

66 activ ity (Go spo daro wic z, Ferrara et al. 1987; Go spo daro wic z, Neufe ld et al. 19 87). Similarly v aried i nter actio ns of p erle c an and F GF-2 with FGFR s hav e be en s ee n in work do ne by other res ear cher s. Kno x et al., 2002, found th at the BaF 3 cell sy st em res pon se w as d ifferen t depe nding n ot only o n the sour ce of th e perle can b ut al so on w hic h FGFR th e cel ls e xpre s sed. Perl ec an ma de by hum an umbi lic al arteria l endo thel ial c ell s (HUAE C) and by a conti nuou s en doth eli al cell lin e (C1 1 STH) stim ulat ed FGF-2- media ted prol iferat ion of BaF3 cell s ex pre ssi ng FGF R-1c. In co n trast, c ell s expr es sin g FGF R- 3 were stimul ate d to prolif erate o nly b y HUAEC p erle can. A naly si s of the gly cos amin ogly can (G AG) ch ain s on th e perle ca n from the se two cel l type s show ed th at the GAG s f rom the HU AEC perl ec an could b e entir ely di ge ste d with h epari n as e, while a portion of t he GAG s on th e C11 STH p erle can w a s re si stan t to hep arina s e. Thes e non-he para n sulf ate G AG s may b e blo c king the d eliv ery of the bound FGF- 2 to FGFR- 3. The finding s of our study are co ns ist e nt with th e phen otyp e of the perl ec an null mo us e, whi ch sh ow s reduc ed Indi an he dge hog expre s sion a nd red uce d prolifer atio n o f chondro cyte s in th e grow th plate (Ari kaw a-Hir as aw a, Wata nab e et al. 1999). In contra st to t he FGFR-3 in BaF3 cell s an d in mo st oth e r cell typ es, FGF R-3 i s a nega tive re gula tor of cho ndroc yte pro l iferatio n in the gro wth pl ate (Colvi n, Bohn e et al. 19 96; Orni tz an d Marie 2 002). That is, w hen the rec eptor in gr owth pl ate chon drocy tes i s ac tiva ted b y bound FGF, it inhibit s Indi an he dge hog ex pre ss ion, and pr olifer ation i s preve nted. In thi s s tudy, w e hav e sh ow n that th e hep aran sulfa te on growth pl ate p erle can bi nd s FGF-2. Ou r data are als o con si ste nt with the chon droitin sulf ate on p erle ca n inhibiti ng FGF-2 d eliv ery to its re cept or. Perlec an in th e growth plate wo uld b e acti ng, a s

67 previou sl y propo se d (Arika wa-Hir as aw a, Wata nab e et al. 19 99), to seq ue ster FGF- 2 aw ay from FGF R-3 an d allow c hondr ocyt e proliferat ion to o ccur. It sh ould b e not ed that a rec ent s tudy (Kv ist, John so n et al. 20 06) sh owe d that th e c hondroit in su lfat e cha in s of perle can c an bi nd to col lag en. This in t eracti on wit h coll age n woul d likely enha nc e the seq ue strati on of the growth fac tor (boun d to perle can) a way from it s re cept or in the growth pla te.

Ackno wledgm ents

This work w a s su pport ed by fund ing fr om Shriner s Ho spit al s for Childr en, North A meric a (to J RH) a nd by NIH gran t HD39 952 (to DMO). W e than k J acki e Ga hag an for t he prep arati on of the manu scrip t.

68 Figure s Figure 5.

1 2 5 Bindi ng of [ I]-FGF-2 to perle ca n in the ca tioni c filtrati on (C AF) 1 2 5 as sa y. [ I]-FGF-2 (0. 086 – 44 nM; 0 . 293 – 150 ng/t ube) w a s adde d to tub es wi thou t () or with per leca n (10 ng G AG/ml; 2 n g GAG/t ube, ). After incub atio n for 1 h, the sa mple s w ere filt ered acro ss t he c ation ic mem bran e and th e c ount s retai ned o n the filt er were d etermi ned. From the sp ecifi c ac tivity th at wa s de termin ed for the lab ele d FGF-2, the n anogr am am ou nts of FGF-2 corre spon ding to the bou nd co unt s wer e cal cul ated an d are sh own. Aft er subtr acti ng ba ckgro und bin ding of FG F-2 to the Zeta Pro be membra ne, the spe cific b indin g is als o indica ted (). The ad dition 1 2 5 of perle can signifi ca ntly in crea s ed [ I]-FGF-2 retenti on at a ll conc entr ation s te st ed (p< 0.04, p aired t-test). All d ata p oint s ha ve error bars re pre sen ting ± S.E.M.

69 Figure 6.

Effect of chon droiti na se or he pariti na s e pretre atme nt of perl eca n on 1 2 5 spe cific b indin g of [ I]-FGF-2 in the CAF a ss ay. Perle can w a s either l eft undi ge ste d (U), or dige sted sep arat ely wit h eith er chondr oitin as e AB C (C) or a mi xture o f hepariti na se s I and II (H). After inc ubat ion of perl ec an (10 ng/ ml ; 2 ng/tub e) for 1 h with 1 2 5 [ I]-FGF-2 (20 ng/ml; 4 ng/t ube), the sampl e s were fil tere d acro s s the ca tioni c membr an e and th e cou nt s retain ed on th e filter w ere determi ned. Equiv ale nt bindi ng of und ige sted and gly co sid as e- treate d perl ec an to the cati onic m embr ane w as confirm ed by immunob lottin g the m embra ne. Hep arit ina se dig es tion of p erle can 1 2 5 signif ica ntly re duc ed [ I]-FGF-2-perl eca n bindi ng (p= 0. 00 3, paired t-t es t). Error bars repre s ent ± S . E.M.

70 Figure 7.

Effect of chon droiti n sulf ate or h epar a n sulfa te on spe cific F GF-2 bindin g in the CAF a s say. Perle ca n (40 ng/ml, 8 ng/tu be) w as incub ate d in tub es c onta ining 0 - 10 00 ng/ml (0 - 200 ng/t ube) chondr oitin sulfa te C ( CSC, ) or bov ine kidn ey HS (). 1 2 5 Followi ng the additi on of 5ng/ ml (1 ng /tube) of [ I]-FGF-2, sam ple s wer e incu bat ed for 1 h, filtere d, and sp ecifi c bind ing of 1 2 5 [ I]-FGF-2 determin ed. R es ults are t he mea ns of d upli cat e determi nati on s.

71 Figure 8.

1 2 5 Bindi ng of [ I]-FGF-2 to perle ca n in the immun opre cipit atio n (IP) 1 2 5 as sa y. Perlec an a nd [ I]-FGF-2 (1 µg/10 6 cpm) w ere mix ed an d aliquo ts c ont ainin g 25-500 n g of perle can w ere immu nopre cipi tat ed with prot ein G be ad s aft er incu batio n i n the ab se nc e of seru m, or with ant is era to p erle can or a ggre can. After wa shi ng to remo ve 1 2 5 1 2 5 unboun d [ I]-FGF-2, the amo unt of [ I]-FGF-2 relea se d from the 1 2 5 bead s by 2 M so dium c hlorid e (A) and the [ I]-FGF-2 remai ning on 1 2 5 the be ad s (B) wa s me as ured. [ I]-FG F-2 elute d by 2 M sodi um chlorid e wa s s ignifi can tly gre ater for perle can v ers us a ggre can anti sera-tr eat ed sampl e s at e ach c onc e ntration of p erle can u s ed (p< 0. 05, paired t-t es t). All data p oint s ha ve error bar s repre s entin g ± S. D.

72 Figure 9.

Effects of c hondr oitin as e and h ep aritin as e pretre atm ent of perl ec an 1 2 5 on [ I]-FGF-2 binding u sin g the IP a ss ay. Perle can (1 25 ng) wa s either l eft undi ge ste d (U), dige sted wit h chondr oitin as e AB C (C), dige ste d with a mi xture of h epari tina s e s I and II (H) or dige ste d with both chon droitin a se an d hep aritin as es ( C+H), in cuba ted w ith 1 2 5 10 5 cpm of [ I]-FGF-2 and imm unopr ecipi tat ed us ing no n-immun e serum or p erle can anti serum and prot ei n G bead s. After w as hing to 1 2 5 1 2 5 remove u nbou nd [ I]-FGF, the amo un t of [ I]-FGF-2 relea sed from the be ad s by 2 M sodi um chl oride was m ea sure d. Hepar itina s e and ch ondroi tina se + hep aritin as e tre at ment s of perle ca n 1 2 5 signif ica ntly re duc ed the amoun t of [ I]-FGF-2 relea se d from the bead s by 2 M so dium c hlorid e (p< 0. 02 , paired t-te st). Error bars repre sent ± S. D.

73 Figure 10.

Bindi ng of FGF2 to solu ble FGF R-1 c a nd FGFR-3 c. Iodin ated F GF2 was add ed to FR AP-co nditi oned m ediu m. Heparin, p erlec an or chondr oitin as e-dig es ted p erle can w a s a lso a dded i n con cen tratio ns ranging from 0 t o 2. 5µg GA G/ml. The recep tors were immunopr eci pitat ed wit h anti- alk alin e phos pha ta se a ntibo dy and Protein G Se phar os e and t he am ount of label ed FGF2 b ound to t he bead s d etermin ed in a g amm a cou nter. All dat a point s h ave error bars re pre sent ing ± S. E. M.

74 Figure 11.

Hepar in and p erle can augm ent ation of [ 3 H]-thymidin e inc orpora tion into FGF-2 tre ate d BaF 3 cel ls. B aF3 c ells expre s sing eith er FGFR- 1c (part A) or FGF R-3c (p art B) wer e t reate d with 20 0 pM F GF-2 and 0–5 µg /ml of eith er hep arin, perl e can, ch ondroit ina se AB C dige ste d perle ca n (perle ca n>pfA B C) or chondroi tina s e AB C dig est ed chondr oitin sulfa te (CS C> pfAB C) an d then rad iola bel ed with [ 3 H]- thymidi ne to d etermi ne prolif erati on rate s. All dat a poin ts h ave error bars re pre sen ting ± S.D.

75 Figure 12

Hypoth eti cal mo del of FGF-2 b indin g t o perle can a nd de livery t o FGF rece ptor. (A) Perlec an wi th hep ar an and chon droitin sulf ate chai ns c an bin d FGF-2 but t he ch ondro itin sulf ate chai ns pr eve nt deliv ery of boun d FGF-2 to th e FGF re cept or. (B) Perlec an with only he para n sulf ate chai ns c an bi nd F GF-2 and d eliv er it to the recep tor.

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Paper II

Fibrobla st Gro wth Factor- 18 Bind s t o Domain I I I of Gro wth Plate Perle can

Simone M-L Smith, Leigh A. W est 1 an d John R. Ha ss ell

Depar tment of Mol ecul ar M edi cine, Un ivers ity of Sout h Florida Coll ege of Medi cin e, Tampa, FL. 1 Current ly at D epartm ent of Chemi ca l Engine ering, Co lleg e of Engine ering, Uni ver sity of Sout h Flori da, Tampa, FL.

Runni ng titl e: FGF-18 b ind s to perl ec a n

Addre s s corre spo nde nce t o: Jo hn R. H a ss ell, Ph. D. De partm ent of Mol ecul ar M edic ine, Uni ver sity of Sou th Florida Coll ege of Med icin e. Tampa, FL. Phone : 813-97 4- 7881. E-mail: jha ss ell@ hea lth. u sf. edu

The abbre viat ion s us ed ar e: C AF, catio nic filtrat ion; C S, chondr oitin sulfa te; D DSH, Silver man- Handm ak er type dys se gmen tal dy spl a sia; D ME M, Dulbe cco’ s modifi ed Eagl e’s medium; DM M B, dimeth ylmet hyl ene bl ue; FGF, fibrobl as t growth factor; FGF R, fibrobl as t growth f actor recept or; GAG, glyco sa minog lyc an; HS, he para n su lfat e; SJS, Sch wart z-J ampe l syndr ome.

83 Abstr act

Fibrobla st gro wth fa ctor-18 (FGF- 18) regul ate s the gro wth plate chon drocy te prolif erati on, hyp ert rophy an d cartil ag e vas cul ariz ation n ec es sar y for endo cho ndral o ss ifica tion. The hepar an s ulfat e prote ogly can p erle can is al so crit ica l for growth plate chon drocy te prolif erati on. FGF-1 8 null mic e exhi bit s kel etal dwarfi sm si milar to th at of perl ec an nu ll mice. Gro wth pla te perle can c ont ain s cho ndroiti n sulf ate ( CS) and h epar an s ulfat e (HS) chai ns a nd FGF-18 i s kn own to bi nd to heparin and to h epar an sulfa te from s ome sour ce s. We u sed ca tionic filtr atio n and immunopr eci pitat ion a s say s to in ve stig ate th e bindi ng of FGF-18 to growth pl ate p erle can. Our d ata sho we d a sat urabl e bind ing wit h a k d of 144 nM. N ear s atura tion, ~1 03 m olec ule s of FGF-18 b ound p er molec ule of p erlec an. Thi s bindi ng wa s not signifi can tly alt ered b y chondr oitin as e nor he pariti na se di ge sti on of perle can, in dic ating that perl ec an GA Gs are not i nvolv ed in FGF-18 bindi ng. FGF-18 bound e qual ly to re combi nant d omai ns I-III of perlecan (Alt 1) expre s sed in COS-7 cell s an d to full-le ngth perl ec an purifi ed from the growt h plat e. Addit ion ally, FGF-18 bound eq uall y to recomb inan t doma in III of perlec an, to Alt1 and to Alt2 (a v ariant of domain s I-III with no h epar an s ulfat e). These d ata i ndic ate t hat low affinit y bindi ng s ite s for FGF-18 a re pres ent in d omai n III of perle can.

84 Introduction

Perlec an i s a larg e (~600 k Da) h epar an sulfat e-co ntai ning proteog lyc an pre se nt in all m amma lian bas emen t membr ane s (Has s ell, Rob ey et al. 1980; Io zzo 2 00 5), in cartila ge (Sun darR aj, Fite et al. 19 95; H andl er, Yurch enc o e t al. 1997) an d in the gro wth plate (G ovin draj, We st e t al. 2002). Pe rleca n is e s sen tial for lon g bone gro wth. The ab s enc e of perl eca n in mice re sul ts in d efe ctiv e endo chon dral o ss ific ation (th e proc es s by which l ong bo ne s grow) during em bryoni c dev elo pment (Ar ika wa-Hir as awa, W ata nab e et al. 1999; C os tell, Gu st afs son et al. 199 9). The chondr ocyt es i n the growth pl ate of p erle can nu ll mic e exh ibit de crea se d prolifer atio n and matri x dep osi tion, whi ch le ad s to sev ere fet al dw arfis m. Silverma n-Ha ndma ker dy s seg ment al dy spl asi a (DDS H), which resul ts from a fu ncti onal n ull mut ation of the perle ca n gen e (Arikaw a-Hira s awa, Wi lco x et al. 200 1 ; Arikaw a-Hira s awa and Yama da 20 01) is a n an alogo us d isor de r in human s. Schw artz- Jam pel syndr ome (S JS) in huma ns i s tho ught t o resul t from produ ction of trunca ted (or red uce d lev el s of full-len gth) perle ca n (Nico le, Davoi ne et al. 2000), with p atie nts sh owing v arying d egre e s of dwarfi sm an d sk elet al ab normal itie s th at mirror tho se of DDS H patie nts b ut are l es s sev ere. Fibrobla st gro wth fa ctor rec ept or-3 (FGFR-3) i s a ce ll surf ac e recep tor expr es s ed in de velo ping gro w th plat e cartil ag e (Wan g, Green et al. 2001). G ain of fun ction m utatio ns in t he hum an FGF R-3 gene cau se d warfi sm s su ch a s a chon dropla sia (Shi ang, Thom pso n et al. 1994), tha nato phori c dys pla si a (Tavormina, R imoin et al. 1995), and hyp och ondrop la sia (W interp ac ht, Hilbert et al. 2000). Activ atin g mutati on s in murin e FGFR- 3 als o cau s e dwarfi sm (N ask i, Colvi n et al. 199 8; Wa ng, Spat z et al. 1999; Iw ata, Ch en et al.

85 2000). Whe n FGF R-3 expr es sio n in the growth pla te of mic e is l ost, there i s incr ea sed chon drocy te prolif er ation a nd hyp ertroph y resul ting in o vergro wth of lon g bone s (Colvi n, Bohn e et al. 19 96; Deng, Wy ns haw- Bori s et a l. 1996). Thi s indi cat es t hat FGF R-3 i s a nega tive re gula tor of cho ndroc yte pro l iferatio n. Fibrobla st gro wth fa ctor s (FGFs) are t he end oge nou s liga nd s for FGFR s. FGFs-2, -9 an d -18 hav e be en sh own to inf luen ce chondr ocyt e prolif eratio n in the d ev el oping gro wth pl ate a nd sub se que ntly influ en ce lon g bon e grow th. The FGF-2 kno ckout mous e ha s no sev ere def ect i n bone l en gth (Mon tero, Ok ada et al. 2000) wh erea s ov er-ex pre ssi on of FGF -2 in the FGF-2 tra ns geni c mous e re sult s in s hort limb s, indi cati n g that FGF-2 c an a ct throu gh FGFR-3 (C offin, Florki ewic z et a l. 19 95; M ancil la, De Lu ca et a l. 1998). Bot h the FGF- 9 tran sge nic mo u se a nd the F GF-18 kno cko ut mous e sh ow sev ere ab normal itie s in lo ng bone gr owth (G arofal o, Kliger-Sp atz et al. 199 9; Liu, Xu et al. 2002; Ohb ay as hi, Shiba yam a et al. 2002), but it i s th e FGF-18 nu ll mous e that exhi bit s a growt h plate p hen otyp e simi lar to th at of the FGFR-3 nu ll mou se (in th e incre as e, altho ugh tra nsi ent, in c hondr ocyt e prolifer ation and in enlarg ed gro wth pl ate s (Liu, Xu et al. 2002; Oh bay a shi, Shib ayam a et al. 2002)). FGF-1 8 ha s a thre e-tier e d importan ce in l ong bo ne growth, affe ctin g chon drog ene si s by s i gnalin g throug h FGF R-3 (David so n, Blan c et a l. 2005), ost eog e nes is by sig nali ng throu gh FGFR-1 a nd/or FGF R-2 (Liu, Xu et a l. 2002; Ohb ay as hi, Shiba yam a et al. 2002), an d growt h plat e va sc ular izati on by reg ulati ng va s cular endot heli al growt h fact or (VEGF) (Liu, Lavine et al. 20 06). FGF-18 is mad e by th e dev elop ing peri cho ndri um, the regio n of conn ect ive tis sue th at surroun ds th e dev elo ping gr owth pl ate (Liu, Xu et al. 2002; Oh bay a shi, Shib ayam a et al. 20 0 2).

86 All FGF s bind to h epari n (As hikari- Ha da, Habu chi e t al. 2004) and he parin h as b ee n sh own to e nha nce the bindi ng of FGF’ s to FGFR’ s (Ornit z and Led er 1992). H epa ran sulf ate (HS), a struc tural anal ogue of h epari n, is pre s ent on gro wth plat e perl ec an at bo th end s (domai ns I a nd V) of the c ore pro tein (Noo nan, Full e et a l. 1991; Tap ana de chop one, H as se ll et al. 1999). We h ave sho wn tha t perle can bi nd s FGF-2 vi a the se HS cha ins a nd ca n medi ate th e deliv ery of FGF-2 to FGF R-1 a nd FGF R-3 (Smith, We st e t al. 2006). FGF-18 is k now n to bind pr efere ntial l y to 2-O-su lfat ed HS (Ashi kari-H ada, H abu chi et al. 2004). The HS on growt h plat e perle can i s 2-O- an d 6-O- sulfat ed (W e st, Govi ndraj e t al. 2006) so, in additi on to bin ding FG F-2, the HS o n perle can mi ght al so bi nd FGF-18. Not al l HS, howe ver, ha s the seq uen ce or sulfat ion pa ttern nec es sar y to bind to F GF-18 (Kn ox, M erry et al. 200 2). In this stud y, we te st FGF-1 8 a s a pot entia l bi nding p artner of gro wth pl ate perle can.

Expe rimenta l Proc edur es

Sourc es of pe rl ecan D NA, per lec an p rotein, gr owth facto r s and antibodie s. cDNA con stru cts enc oding for p erle ca n domain I-III and for do main III were obta ined from pr evio us w ork (Cha krav arti, Horch ar et al. 1995; Do lan, Hor char e t al. 1997). Lar ge-s cal e pla smi d prep s wer e prepar ed u sing th e Endo-fre e Pla smid Max i Kit (QIAGE N) and con struct s w ere ver ified u sin g res tricti on endo nucl ea se s. Rec ombin ant h uman fibr obla st gro wth factor-18 (F GF-18) wa s purch as ed from PeproT ech. Purifi ed bo vine gro wth pl ate p erlec an

87 (Govindr aj, We st et al. 2002) a nd anti mous e perl ec an anti body Ab378 (W e st, Govin draj et al. 2006) w ere prep ared as pr eviou sl y des crib ed.

Tran sfe ction. High glu co se (4. 5 g/L) D MEM (Fi sher Scient ific) w as supp lem ente d to 10% fetal b ovin e s erum, 1% antib iot ics (p enic illin- stre ptomy cin) and 2% glut amin e and u s ed a s our gro wth medi um to cu lture COS-7 cell s in 100 mm cell- cultur e di she s (Fi sher S cient ific). Lipofect amin e 2000 (L20 00, Invitrog e n) was u s ed to tran sfe ct conflu ent C OS-7 c ell s ac cordin g to the manufa cturer’ s dire ction s. Briefly, th e cel ls w ere rin se d with seru m- and anti bioti c-free gro wth medium (our “tran sfe ctio n medi um”). For the con stru ct s Alt1 (domain s I-III) and Alt2 (d omai ns I-III made wit hout h epar an sulfa te), cel ls w ere tra nsf ect ed wit h 12 µg of DNA us ing 45 µl of L2000 in 15 ml of tra nsf ecti on med ium . For the domain III con struct, c ell s wer e tran sfe cted with 18 µg of DNA u sin g 45 µl of

L2000. The cel ls w ere c ulture d at 37º C /5% CO 2 for 5h, then cha nge d to growth m edium for a noth er 16-18h. The tran sfec ted c ell s wer e then furth er cult ured in 15 m l of fresh transf ecti on me dium an d harve st ed after 4 8h.

Purifying R ecombin ant Product s. The 48h me dia from tra nsf ect ed C OS-7 cell s were r econ sti tute d to 4 M gua nidin e H Cl by the addit ion of 0. 5 g of guanid ine H Cl/m l of media. The m edia were t hen spin c onc entrat ed to le s s tha n 2 ml and fraction ate d on 4 M g uani din e HCl- equ ilibrat ed Sup eros e 6 col umn s (GE Healt hcar e). Forty 3 ml fracti on s (1. 2 column v olum es) w ere colle cte d, then 1 µl of e ach fra ctio n w as do tted u nto nitro cel lulo se membra ne (Bi o-R ad) and imm unob lott e d for perlec an u sing Ab378.

88 Perlec an-po sit ive fra ction s w ere di alyz ed ag ain st di still ed wa ter an d 18 µl aliqu ots of t he frac tion s wer e ru n on SDS-PAGE. Frac tion s from the Alt1 colum n run wer e dige st e d with 10 mU of pro tea s e-free chondr oitin as e AB C a nd 5 mU e ach of hepar atin as e s I and II (As soci ate s of C ap e Cod) b efore SDS- PAGE, as d es cribe d bel ow. The DIII and Alt 2 fracti ons were l eft u ndige st ed si nc e the se recomb inan t produ ct s hav e no GA Gs at tach ed. Fract ion s det ermin ed by We ster n blot to c onta in the re com bi nant prod uct b ut non e of the endog eno us COS-7 c ell p erlec an w ere poole d and th e amo unt of perle can e sti mat ed by dir ect c ompari so ns wit h know n amou nt s of EHS perle can in th e dim ethyl meth ylen e blue (D M MB) a s say (Farndal e, Butt le et a l. 1986) an d in W est ern blot s.

Glyco sida se Dige stion of Pe rle can G AGs. Where i ndic ate d in the re su lts, p erlec a n was eith er sh am dig est ed, dige ste d with 10 m U of cho ndroiti na se ABC or di ge ste d with 5 mU eac h of hep aritin as es I a nd II before b eing u sed i n the C AF or the IP as sa ys. Dig e stion s w ere don e in 100 µl of diges tion buff er (20 mM Tris HCl, 5 m M cal cium chlorid e an d 0 .2 mg/ml prote as e-free BSA, pH 7. 4) at 37° C for 3h.

Radiolab ellin g of gro wth factor. 1 2 5 Sodium Iodid e (0. 5 mCi, Perk inElm er) activ ate d in IODO- GEN- coat ed tub es (Pi erc e) wa s us ed to rad i olab el 5 µg of FGF-18 u sin g Pierce’ s s uppli ed prot ocol ( Chi zzon ite indirect m etho d) as previou sl y de scrib ed for FGF-2 (Smit h , West e t al. 2006). The iodina ted FGF- 18 wa s purifi ed by chro matogr aphy o n Hep arin Sephar os e 6 Fa st Flow b ead s (GE He al thcar e) and el utio n with 2 M NaC l. The purifie d FGF-18 w as di aly z ed ag ain st PBS a nd the

89 spe cific acti vity of th e 1 2 5 I-FGF-18 det ermine d as b efore (Smit h, We st et al. 20 06).

Cationi c Filtration A s say. The bindin g of 1 2 5 I-FGF-18 to perl ec an was d etermi ned u sin g a catio nic filtr ation ( CAF) a s say a s pre v iousl y de scri bed (For ste n, Wang et al. 2000 ; Smith, We st e t al. 2 006). The bindi ng buffer w a s 0. 05 M Tris H Cl, 0. 15 M Na Cl and 2 m g/ml prote a se-fre e BSA, pH 8. 0. Briefly, iodi nat ed FGF-18 wa s inc ubat ed with or wi thou t perle can in b indin g buffer at ro om tem peratur e for 1h in a fin al incub atio n volum e of 200 µl. The mixt ure wa s the n filtere d throug h catio nic Zet a-Prob e membr ane (B io-R a d) by vacu um filtrat ion u sing a Minif old I 96-w ell app arat us (Sc hlei cher an d Schu ell, Ke en e, NH, USA). The well s w ere rin sed t o remov e unbou nd 1 2 5 I-FGF-18, the membra ne w as dri ed an d the w ell s ex ci sed and co unt ed in a g amma count er. For the initi al do se-r es pon se CAF a s say, a satur atio n bindin g curv e and a S cat chard p lot of t he dat a wer e gen erat ed u sing the Sigm aPlot R egr es sio n Wiz ard® (Si gmaPlot 8. 0). To study th e effe ct of GA Gs o n FGF-1 8-perle can b indin g, perle can (full-l engt h or Alt1) w as di ge ste d with c hondro itin as e AB C or heparit ina se s I and II bef ore bei ng use d in the CAF a s say. On ly full-leng th perl ec an and r ecom bina nt d omain I-III produc t Alt1 w ere use d in the CAF a s say sin ce th e cat ion ic Zeta-Prob e membr ane capt ure s neg ativ ely ch arge d mole cul es. Reco mbin ant prod uct s ma de withou t GAG c hai ns (th e Dom ain III a nd the Alt 2 produ ct s) coul d be eval uat ed for FGF-18 bi nding o nly by an immun opre cipit ation as sa y.

Immunopr ecip itation A s say. The bindin g of FGF-18 to p erle can w a s studi ed u sing a n immunopr eci pitat ion (IP) as s ay a s prev iousl y de scri bed (Kn ox,

90 Merry e t al. 2002; Sm ith, We st et al. 2006). Purified re combi nan t perle can w a s reco nst itute d to 0.2 ml in IP buffer (1% Triton X-100, 20 mM Tris H Cl, 0. 15 M Na Cl, pH 7. 4 ). 1 2 5 I-FGF-18 was ad ded and the s ampl es w ere in cub ate d at room te mperat ure for 1h wit h mixing. Five micro liter s of eit her ant i- mou se perle can a ntibo dy Ab 378 (or 5 µl of pre-immun e rabbi t ser um a s cont rol) were ad ded and th e sam ple s inc ubat ed at 4 °C for 2h. Aft er additio n of 20 µl of Protein G Seph aros e be ad s (GE Hea lthc are) fo r 2h at 4ºC, the samp le s wer e centrifu ged for 5 min at 250 0xg, the b ead s wa sh ed thre e time s wi th IP buffer and th e radio acti vity in th e p ellet s or in 2 M elua te s mea sure d in a gam ma co unter. Perl ec a n was al so eith er sh am- dige ste d or dige st ed with pr ote as e-free chondroi tina s e AB C an d hepar atin as e s I and II before b eing u se d in the IP a ss ay.

Denatur ation and r educti on/alky latio n of the perle can co re protein. To determin e if FGF-18: perl eca n bindi ng wa s dep end ent on core protein stru cture, w e us ed FGF-18 i n a CAF a ss ay wit h purifie d perle can th at w as e ither u ntrea ted, or dena tured w ith 8 M ur ea or both de natur ed an d redu ced/ alk ylat ed with ure a and 2 5 mM DTT (GE Heal thc are) / 50 m M Iodoc aet amid e (Pierce). The p erle can w as dialy zed agai nst d ist illed wat er after t reatme nt an d before u s e in the as sa y.

Statistic s. All dat a are e xpre s sed a s th e mea n of four replic ate s +/- sta ndard error of the me an. Stati sti cal c omp aris ons w ere ma de u sing Studen t’s t-te st, wh ere a ppli cabl e. P<0 . 05 was con sid ered signif ica nt.

Res ults

Binding of FGF-18 to full-len gth perl ecan in the C AF as say. 1 2 5 I-FGF-18 (33 – 2385 ng /ml; 0. 75 – 109 nM) w a s incu bat ed wit h or without 2 n g (in DM M B GA G cont e nt, therefor e 10 ng of tot al perle can)/m l of perle ca n purifie d from the growth p lat e. The sam ple s wer e filter ed over a cati onic membra ne an d radiol iga nd bindin g to the m embran e wa s d etermi n ed us ing a g amma count er. Count s from in cub ation s wit hout p erle can w ere s ubtra cte d from thos e with p erle can at equ ival ent FGF- 18 con centr atio ns to determi ne th e sp ecifi c bin ding. The n a nomolar amoun ts of FGF-1 8 corre spon ding to th e bou nd co unt s wer e cal cula ted from th e sp ec ific activ ity of the l abe led FGF- 18 and are sho wn (Fig. 13). The dat a sho w that th e bind ing of FGF-1 8 to per leca n wa s s atur able abov e

84.5 nM FGF- 18 add ed, with a di sso ci ation c on stan t (k d ) of 145 nM as d etermi ned from Sig maPlo t Scat cha rd analy si s.

Effect of Gly co sida se Tre atment of Perle can on FGF-18 B inding in the CAF and IP a ss ays. Growth pl ate p erle can cont ain s both C S and HS ch ain s (Govi ndraj, We st et al. 20 02). Purified gro wth pl at e perle can (1 0 ng GA G/ml) was fir st s ham-d ige st ed or dig est ed wi th chon droitin as e A BC or with he pariti na se I and II bef ore inc ub ation wi th 1 2 5 I-FGF-18 (80 ng/ml) in the CAF a s sa y. Contro l bindi ng (no perl eca n) wa s subtr act ed from tot al bindi ng (with p er leca n) to giv e sp ecifi c bindin g sho wn. We fo und th at neit her chondr oitin as e (C) nor hepari tina s e (H) treatm ent of per lec an signif ica ntly aff ect ed the bindin g of perle can t o FGF-18 in c omp ariso n to sh am-di ge ste d

92 perle can ( sham, Fig. 14). W e al so u sed the IP as sa y to det ermin e if 1 2 5 I-FGF-18 could b ind to p erle can wi t hout int act G AG ch ain s pres ent. Purifie d perl eca n (30 ng G AG /ml) wa s eith er sh am dig es ted (sham, Fig. 15) or dig e sted with c hond roitina se ( C) or hep aritin as es I and II (H) or a mixture of bot h (C+ H ). The perlec an wa s th en incub ate d with 1 2 5 I-FGF-18 (40 n g/ml) and s erum (pre-imm une or anti-p erlec an), ca pture d with Prot ein G bead s a nd the F GF-18 bindin g det ermine d. There wa s signifi c antly mor e bindi ng of 1 2 5 I- FGF-18 to th e bea ds w ith an ti-perl ec an serum th an wit h pre-immu ne serum. Ther e wa s no signifi ca nt differ enc e, howe ver, whe ther th e perle can u se d had it s G AG ch ain s int a ct (sh am trea tment) or h ad only on e type of G AG (C or H tr eatm e nt) or had no int act G AG s (C+H tr eatm ent).

Effect of Co re Prot ein D enaturat ion and Reducti on/Alk ylation on 1 2 5 I-FGF-18 Binding. To determin e if core pr otei n stru cture has an effe ct on FGF- 18:perl ec an bin ding, we u s ed 50 ng of FGF-18 in a C AF a ss ay wi th 2 ng GAG of p erle can th at wa s ei ther u ntreat ed, or den ature d with 8 M urea or b oth de natur ed an d redu ced / alkyl ated with ure a an d 50 mM DTT/ 100 m M Iodo cae tamid e. The binding of FGF- 18 to perle can w a s redu ced by m ore tha n 50 % when the core pro tein w a s struc ture w as alter ed by red ucti on an d alkyl ation (Fig ure 16). The decre a se w as l es s dra sti c but still sign ificant when t he cor e prot ein was d en ature d only.

Binding of 1 2 5 I-FGF-18 to full lengt h perle can and to Alt1 in the CAF as say. For Fig. 17, the bindi ng of 1 2 5 I-FGF-1 8 to full-len gth perl ec an wa s comp ared to th at of AltI, a re combi nan t product cont ainin g only th e

93 N-termin al dom ain s I-III of perlec an ( Dolan, H orch ar et al. 199 7). At con centr atio ns w ell be low satur atio n (as de termin ed in Fig. 13), 1 2 5 I-FGF-18 (20 ng/ml in Fig. 17 A, 16 0 ng/ml in Fig. 17 B) wa s incub ate d with full-l engt h perl ec an or recomb inan t Alt1 (10 n g GAG/ml). B oth p erlec an prod uct s ha d simil ar bindi ng ca pa city (~50%) for 1 2 5 I-FGF-18 at both F GF-1 8 conc entr ation s u sed.

Binding of 1 2 5 I-FGF-18 to pe rle can d omains I-I I I and to domain II I in the IP as say. The domai n in the N-t ermin al half of p erlec an th at bind s FGF-1 8 was d et ermine d in the IP a s say b y incu bating 4 0 ng/ml of 1 2 5 I-FGF- 18 witho ut perl ec an, or with 15 0 ng/ml of a recombi nant pr odu ct for domain III of perl ec an (Ch akra varti, H orchar et al. 1995) or wit h Alt1 or with A lt2, a rec ombin ant pro du ct of domai ns I-III mad e withou t GAG s (Dol an, Horc har et al. 1997). FGF-18 bou nd eq uall y to domai n III as it bou nd to th e doma i n I-III products (Fig. 18).

Dis cus sio n

In this study we u sed a cati oni c filtrati on (CAF) a s say a nd an immunopr eci pitat ion (IP) as s ay to ide n tify FGF-18 a s a no vel bindin g partn er of growth pl ate p erle c an. FGF-18 bo und to n ativ e perle can p urified from th e dev elo ping growth pl ate (Fig. 13). Remo val of th e cho ndroiti n su lfate ( C S) and hep aran sulf ate (HS) chai ns of p erlec an di d not si gnifi cant l y reduc e FGF-18 bi ndin g (Figs. 14 an d 15). Thes e da ta s how th a t no other gro wth pl ate molec ule s are n ee ded to m edi ate th e in teract ion b etwe en p erlec an and FGF-1 8 and th at the b indin g is to t he core pr otei n of perle can.

94 In further confirm atio n of thi s finding, denatur ing an d reduci ng/a lkyl ating t he cor e prote in of perlec an s ignifi can tly decre a sed th e bind ing of FGF-1 8 to pe rleca n (Fig. 16). With th e compl ete tre atm ent (ure a for den aturat ion, DTT for reductio n and Iodoa cet amide for a lkyl ation), th e bin ding of FGF-18 t o perle ca n was r edu ced c los e to ba ckgr ound (bi nd ing of FGF-18 to th e C AF as sa y membr ane a lone). FGF-1 8 al so b ound eq uall y well t o the recomb inan t produ ct s Alt1, Alt2 and D III of perleca n mad e in CO S- 7 cell s (Fig s. 17 and 1 8). Thes e recom binant pr odu cts are of the expe cte d si ze, co nformati on an d GAG cont ent, a s se en in W e stern blots of t he vari ou s produ ct s unred uce d, reduce d or treat ed wit h GAG ly as es (d ata n ot sh own). Purifi ed full-lengt h perle ca n from the growth pl ate c ont ain s 25% of its t otal GAG s a s HS and 7 5% as CS, recomb inan t Alt1 c onta in s 60% HS an d 40% CS, and rec ombin ant Alt2 do es no t cont ain G AG s, yet th ey all bind FG F-18 com par ably. This s ugge st s th at neit her th e inta ct G AGs o n perl eca n nor the G AG stub s aft er glyc os ida se di ge stio n influ enc e FGF-18 bi nding. The Alt1 produ ct i s the N-t ermin al third of domain I co ntain ing th e serin e GA G att achm ent site s plu s al l o f domain s II and III (Dola n, Horch ar et al. 199 7). The Alt2 prod uct is mad e witho ut GA Gs sin ce the s erine s in d omain I th at are G AG a ttach ment sit es w ere mut ate d to threoni ne s (Dol an, Horc har et a l. 19 97). Domain III al so bo und equa lly we ll to FGF-18 as A lt1 an d Alt 2 did, whic h pinpo int s the bindin g sit e of FGF-18 to d omain III o f perlec an (Fig. 18). The GAG o n perle ca n con stit ute s 20% of its mol ecul ar ma ss (Dolan, Hor char et al. 1997 ; Tapan ad e chopo ne, Ha s sel l et al. 199 9; Govindr aj, We st e t al. 2002). Theref or e, the 2 ng/t ube (10 n g/ml) of perle can G AG u sed i n our initi al do se- resp ons e C AF a ss ay (Fig. 13) corre spon ds to 1 0 ng of perl ec an. The molec ular ma s se s of perl ec an and FGF-1 8 are ~6 00 kD a (Smith, We s t et al. 2006) a nd 22 kD a

95 (Peprote ch prod uct d ata she et) re spe cti vely. A s det ermine d from our sat uratio n bindi ng curv e s hown i n Figu re 13, near satur ation (i. e. at 85.4 nM FGF- 18 add ed) our c alc ulati o ns yi eld a stoic hiom etry of ~103 FGF-1 8 mole cul es b ound p er mol ecul e of perl eca n. This sugg e sts t hat gro wth pl ate p erlec an i s cap able of servi ng a s a reser voir for larg e amou nt s of FGF-18, a functio n previ ous ly s hown for perlec an wit h oth er growth fa ctor s (Iozzo 19 94; M ongi at, Taylor et al. 2000 ; Smith, We st e t al. 2006). Scat chard anal ysi s of th e bindin g dat a usi ng Sigm aPlot show ed a k d of 144 nM for the intera ction b etw ee n FGF-18 a nd perl ec an (Figure 1 3). Thus, growt h plate p erle can i s a lo w affinit y bindi ng partner for FGF-18 (Y ayo n, Klag sbru n et al. 199 1; Fay ein, Co urtoi s et al. 19 92; M os cat elli 1992). FGF-18 bou nd to dom ain III of perle ca n. Domain III ha s a calc ulat ed mol ecu lar ma s s of 120-13 5 kDa (No onan, Ful le et a l. 1991), ac coun ting for ~2 5% of the ma s s of total core pro tein, an d is organi zed in to thre e ide ntic al s ubdom a ins th at sh ow e xten siv e tertiary struc ture. Eac h of the thr ee s u bdomai ns con si sts of a 28-41 amino a cid c yst ein e-rich re pe at follow ed by a 19 2-199 a mino a cid cys tein e-free g lobul ar dom ain follo we d by three additi ona l cy stei ne- rich repe at s (tota ling 14 7-168 a mino a cid s) (Noon an, Fulle et al. 1991). Dom ain III sho ws sub st anti al h omology t o the short arm of the lami nin A c hain, e ven sho wing th e sam e vari abilit y in the number s of cy st eine re si due s per re pea t and the sam e R GDS ami no acid seq uen ce in th e s eco nd glo bular d omain (No ona n, Fulle et al. 1991); thi s s equ enc e i s acti ve in int egr in bindin g and m edia te s the cell-a dhe si ve prop ertie s of perl ec an (C hakra varti, Hor char et al. 1995; Ioz zo 20 05; Kn ox an d Whit eloc k 2006). The dis ulfid e-bond ed cys tein e-rich re pe ats of d omai n III are homologo us t o epid ermal growth fa ctor (EGF) and confer a r esi s tanc e to prot eoly si s

96 (Chakr avart i, Horch ar et al. 199 5). We can s pec ulat e, then, th at a growth fa ctor (s uch a s FGF-1 8) boun d to this sta ble do main III might be mor e effe ctiv ely prot ect ed in the extra cel lular m atrix. The repet itive stru cture of d omain III (which w ould pro vide multipl e bindi ng si te s) and th e ten den c y of fibrobla st gro wth fac tors to multimer ize (H armer, R obin son et a l. 2006) may e xplai n the 143:1 stoi chiom etry of the F GF-18:p er leca n inter acti on; th e bindi ng of FGF-18 to perl ec an co uld en han ce F GF-18 multim eriz atio n. Domai n III is alre ady kn own to b ind to variou s prote ins i nclu ding integrin s, FGF-7, FGF- BP (FGF bind in g protein), PDG F (plat elet derive d growt h factor) a nd WA RP (von Willebr and fa ctor A dom ain- relate d prote in, whic h is expre s sed i n chondr ocyt e s and aff ect s EC M struc ture) (C hakr avarti, Hor ch ar et al. 1995; Go hring, Sa sa ki et al. 1998; Mongi at, Taylor e t al. 2000 ; Mo ngiat, Ott o et al. 200 1; All en, Bat eman et al. 200 6). We hav e s hown i n this stud y that do main III als o bind s FGF-18. FGF-2 is a noth er low affin ity bin ding partner of p erle can a nd bind s exc lu sivel y via p erle can’ s HS c h ain s (Avie zer, Levy et al. 1994; Kn ox, M erry et al. 20 02; Smith, We st et al. 20 06). Perle can in the mou se mo del h spg 2 ∆ 3 / ∆ 3 l ack s th e HS atta chm ent s ite s in dom ain I but is st ill s ynth esi ze d as a HSPG b e cau se of th e HS att ach ment site i n domai n V. The h spg2 ∆ 3 / ∆ 3 mou s e sho ws d efe ctiv e len s c aps ule formation ( Ro ssi, Morita et al. 200 3), dela yed w ound h eali ng an d impaire d angi oge ne si s (Zhou, Wan g et al. 2004), incr ea sed smoo th mus cle c ell prolif erati on (Tran, Tran-Lundmark et al. 200 4) and abnorm al glom erular filtr atio n (Morit a , Yoshim ura et a l. 2005) but no sig nific ant c artila ge or bon e def ect s. This sugg es ts e ither th at the si ngle HS cha in on dom ain V i s s uf ficient for gro wth fa ctor (i. e. FGF-2) acti on in th e growth p lat e or th at, as th e norma l-len gth bone s of the F GF-2 kno ckout i ndic ate (Mont ero, Oka da et al. 2000),

97 FGF-2 is no t a cru cial lig and for FGF R -3 and, thu s, for endo chon dral o ss ific ation in t he grow t h plate. In co ntra st, the FG F- 18 knoc kout h as d efe cts i n end ocho ndr al os sifi cati on (Liu, Xu et al. 2002; Oh bay a shi, Shib ayam a et al. 20 0 2) which sugg e sts t hat FGF- 18 is th e cruc ial FGF in l ong bo ne gro wth. FGF-18 bin ds t o the c ore protein of p erle can. The c ore prot ein o f perlec an i s pre sent i n the hspg 2 ∆ 3 / ∆ 3 mo us e, whic h ha s norma l en docho ndral o s sific atio n but i s abs ent in th e perl ec an null m ou se, whi ch ha s def ecti ve en doc hondr al os sific ation (Ar ika wa-Hir as aw a, Wat a nabe et al. 1999 ; Co ste ll, Gust af sso n et al. 19 99). We prop os e th en, from our finding s, tha t the cor e protei n of perle ca n may b e an important m edi ator of FGF- 18 acti on.

98 Figure s

Figure 13.

1 2 5 Saturati on bin ding of I-FGF-18 to g rowth pla te perl ec an in th e 1 2 5 catio nic filtr ation ( CAF) a s say. I-FGF-18 (33 – 238 5 ng/ml; 0. 7 5 – 109 nM) w a s add ed to tub e s witho ut or with perl ec an (8 ng GAG/ml). After in cub atio n, the s ampl es w ere filt ered a cro ss th e catio nic m embran e an d the c ount s ret ai ned on th e membr ane were determi ned. Spe cific ally b ound FG F-1 8 (spe cific count s [tot al count s min us c ount s bou nd wit hout p er leca n] divid ed by spe cific activ ity an d expr es se d a s nan omolar) i s sh own in t he s atur ation bindin g curv e. Scat chard anal ysi s (in s et) of the bin ding d ata g ave a dis soc iati on con st ant (k d ) of 145 n M. Error bars repre s ent ± S. E.M.

99 Figure 14.

Effect of chon droiti na se or he pariti na s e pretre atme nt of growt h 1 2 5 plate p erle can o n I-FGF-18 bind ing usin g the C AF a ss ay. Perlec an w as either sh am dige st ed (s ha m), or digest ed s ep arate ly with eit her ch ondroit ina se AB C (C) or a mixture of h epari tina s es I 1 2 5 and II (H). After incub atio n of perle c an (10 ng G AG/ml) wi th I- FGF-18 (80 ng/m l) for 1h the samp le s were filt ered a cro ss t he catio nic m embran e an d the c ount s ret ai ned on th e filter w ere determi ned. For e ach tr eatm ent, co ntro l value s (no p erle can) w ere subtr act ed from ex perim enta l valu e s (with perl ec an) to giv e our resul ts. N eith er chon droitin a se nor h ep aritina s e dige sti on of 1 2 5 perle can signif ica ntly c han ged I-FG F-18-perle ca n bindi ng. Error bars re pre sent ± S. E. M.

100 Figure 15.

Effects of c hondr oitin as e and h ep aritin as e pretre atm ent of perl ec an 1 2 5 on I-FGF-18 bindi ng u sing th e imm unopre cipit atio n (IP) as say. Perlec an (30 ng GAG/ ml) wa s eit her s h am dige st ed (s ham), dig est ed with ch ondroit ina se AB C (C), dige st ed with a mixtur e of hepari tina s es I an d II (H) or diges ted with both chon droitin a se an d 1 2 5 hepari tina s es (C +H), inc uba ted wi th I-FGF-18 (40 ng/ml) a nd immunopr eci pitat ed u sing pr e-immu ne serum or p erle can anti serum 1 2 5 and prot ein G b ead s. After wa sh ing to remove un boun d I-FGF- 1 2 5 18, the amo unt of I-FGF-18 rel ea se d from the bea ds b y 2 M N aCl was m ea sur ed. For ea ch trea tmen t, con trol valu es (n o perle ca n) were subtr act ed from ex perim enta l valu e s (with perl ec an) to giv e our resul ts. C hondro itin as e, hep aritin as e, and ch ondroi tina se + hepari tina s e treatm ent s of perl ec an did not sign ific antly c han ge th e 1 2 5 amount of I-FGF-18 r ele as ed from t he be ad s by 2 M N aCl. Error bars re pre sent ± S. E. M. * denot e s sig n ifican ce c ompar ed to pre- immune control (p < 0. 05). 101 Figure 16

Effect of growth pl ate p erle can core p rotein d enat uratio n and reduct ion/ alkyl atio n on 1 2 5 I-FGF-18 bi nding. To det ermin e if core protein stru cture h as an effe ct on FGF- 18:perl ec an bin ding, we u s ed 50 ng of FGF-18 in a CAF a s sa y with 2 ng GAG of p erle can th at wa s either u ntrea ted, or de natur ed wit h 8 M urea or b oth de natur ed an d reduc ed/ alkyl ate d with ure a an d 50 m M DTT/ 100 m M Iodoa cet amide. The b indin g of FGF-18 to perlec an w as re duc ed by more tha n 50 % when t he cor e prote in was stru cture w a s alt ered by reduct ion an d alk ylati on (Figur e 16). The decr ea se w as l e ss dr ast ic but stil l sig nific ant w hen th e cor e prot ein wa s de nat ured on ly.

102 Figure 17.

1 2 5 Bindi ng of I-FGF-18 to na tive ful l-l ength p erle can and to 1 2 5 recomb inan t perl eca n doma in s I-III in the C AF as s ay. I-FGF-18 (Figure 17 A: 20 ng/m l, Figure 17 B: 16 0 ng/ml) wa s in cub ated w ith no perle ca n (-) or with 10 ng GA G/ml of either full-l eng th perl ec an purified from th e growt h plat e or Alt1 (recombi nant d omai ns I-III). After 1h, the sam ple s wer e filter ed acr os s the c atio nic m embran e and the coun ts ret ain ed on th e filter w ere det ermin ed an d divid ed by the s pec ific a ctivit y to det ermin e ng b ound. For both p erle can prepar ation s, ther e wer e simi lar per cen tage s (~ 50%) of FGF-18 bound a t both th e low (Fig ure 17A) an d high (Figur e 17B) conc entr ation s of FGF-18 u s ed. Error bars re pre sent ± S.E.M. * denot es sig nific anc e com pare d to con trol with no p erle can (p < 0. 05).

103 Figure 18.

1 2 5 Bindi ng of I-FGF-18 to differ ent p e rleca n doma ins i n the immunopr eci pitat ion (IP) as s ay. Re co mbinan t perle ca n doma in III (DIII), domains I-III (Alt1) a nd dom ai ns I-III with no GA G s (Alt2), 1 2 5 eac h at 150 n g/ml, wer e mixe d se para t ely with I-FGF-1 8 (40 ng/ml) and t he per lec an w as imm unopr ecipi tat ed with Prot ein G bead s aft er incu bati on wit h pre-immu n e seru m, or with ant is erum to perle can. A c ontrol w as add ed wh ere n o perle can w a s us ed in th e 1 2 5 incub atio n (-). After was hing to r emov e unbou nd I-FGF-18, the 1 2 5 1 2 5 amount of I-FGF-18 r emai ning on t h e bea ds w as d et ermine d. I- FGF-18 bou nd to th e bea ds w a s sig nifi cantl y grea ter u sing a nti- perle can serum v ers us pr e-immu ne s er um (p< 0. 05, pair ed t-te st). There wer e no s ignifi cant d ifferen ce s, howe ver, amon g the thr ee type s of perl eca n that were u se d. Error bars repr es ent ± S. E.M. * denot es sig nific anc e com pare d to con trol with no p erle can (p < 0. 05).

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Yayo n, A. , M. Klag sbrun, et al. (1991) . "Cell surfa ce, he parin-l ike molec ule s are r equire d for bindi ng of bas ic fibrob la st growt h factor to it s hig h affinity r ece ptor. " Ce ll 64(4): 841-8.

Zhou, Z. , J. Wang, et al. (2004). "Im pa ired ang ioge ne si s, dela yed wound h eali ng an d retard ed tum or gro wth in perl ec an he para n sulfa te-d efici ent mi ce. " Can cer R e s 64 (14): 4699-7 02.

109

Paper III

Histon e H3 is a bindi ng partn er of p erle can in the de vel oping growth plat e matrix

Simone M-L Smith 1 , Pras anth i Govi ndr aj 2 , Leigh We st 2 a nd J ohn R Has s ell 1

Depar tment of Mol ecul ar M edi cine 1 , C olleg e of Me dici ne, Univer sit y of South Flori da, Tamp a, Fl, 33612 and Cent er for Re se arch in Sk ele tal D evel opm ent an d Pediatri c Orth oped ic s 2 , Shriner s Ho spit als for Childr en – Tam pa, Fl.

Runni ng Title: His tone H3 bind ing to perle can

Abbrev iati ons u s ed: 1 2 5 I, Iodine-12 5; CAF, ca tioni c filtrati on a ss ay; CS, chon droitin sulf ate; D DSH, dy s seg menta l dys pla si a, Silverm an- Handm ak er type; E CM, ex trac ellul ar m atrix; GA G, glyco sa minog lyc an; H DA C, histo ne d e acet yla se ; HS, hep aran sulfa te; HSP G, hep aran sulfat e prot eog lyca n; SJS, Sch wart z-J amp el syndr ome.

Corre spo nding author: John R. Ha ss ell, PhD Depar tment of Mol ecul ar M edi cine, C o llege of Med icin e, Univer sit y of South Flori da 12901 Bruc e B Do wn s Blv d., MDC Bo x 7 Tampa, FL 33612

Support ed by gra nts from th e Shrin ers Hos pital s for Ch ildre n (to JR H).

110 Abstr act

Perlec an i s the m ajor he para n sulf ate p roteogl yca n (HSPG) in growth pl ate c artil age. It s core pro tein and att ach ed he para n sulf ate chai ns m ediat e int erac tion s with m any diver se prot ein s. Perle can i s es se ntial for pro per s kel etal d eve lopm ent, es pe ciall y in long b one s. The aim of thi s st udy w as t o ident ify p erlec an-bi nding pr otei ns th at are end oge nou s to th e growth p lat e. Affinity chro mato graph y isol ated h ist one H 3 a s a perl ec an-bin di ng protei n from the chondr ocyt e s urfac e/matr ix. Liquid ph as e (cati onic fil tratio n) bindin g as s ays confirm ed th e inter acti on as sp ecifi c, and de pen dent primarily o n HS ch ain s of perle ca n, alt hough c hondr oitin sulfa te chai ns a nd the p erle can core w ere al so involve d. Immunohi sto che mis try al so s how ed s ta ining for perl ec an an d hist one H3 in growt h plat e cart ilag e with a not icea ble c olo cali zati on of the two prote in s. Thes e res ult s ca n help u s better un der stan d the gro wth factor ind ep end ent co ntrol th at perl eca n exert s to fa cilit ate lon g bone gro wth.

Keyw ords : perl eca n, growth pl ate, hi st one.

111 Introdu ction

The hep aran sulfat e prot eogl yca n (HSP G) perle can h as a protein core of fiv e dist inct, hi ghly c o nser ved do main s (I-V) with glyco sa minog lyc an (GA G) cha in s atta c hed to dom ain s I and V (Iozzo 19 94; Ioz zo, Co hen e t al. 1994 ; Smith and H as s ell 200 6). Domai n I was fir st co ns ider ed uniq ue t o perle can (N oona n, Fulle e t al. 1991) but w a s later sho wn to co ntai n an SEA modul e import ant for glyco syl ation of th e GA G att achm e nt sit es in D omai n I (Bork and Patt hy 199 5). Domai ns II to V, ho wever, h ave ho molog y to motifs in epid ermal gro wth fa ctor, lam inin, the lo w den sity lipoprot ein re cept or and N eura l Cel l A dhe sion Mol ecul e (Noo nan, Fulle et al. 1991). Perle ca n is a compo nent of all b a sem ent membra ne s and h as b ee n sh own to in te ract wit h other c ompo nent s found in ba s emen t membr ane, in cludi n g laminin, ni dog en, coll age n II and fibrone ctin (Io zzo 19 94; Ioz zo, Coh en et al. 19 94). Perle can has als o be en sh own to b ind c ertai n growth fa ctor s su ch a s FGF-2 (Avie zer, He cht et a l. 1994) an d FGF- 7 (Mongi at, Taylor e t al. 2000) an d is kn own to b e upre gula ted by TGF-β (Ioz zo, Pillari se tti et al. 1997). Perlec an i s al so pre se nt in c artila ge an d in the grow th pla te (Sundar Raj, Fite et al. 199 5; Ha ndler, Yurch enc o et al. 199 7; French, Smit h et al. 199 9). The growth plate i s a s pec iali zed r egion of cartil age th at reg ulat es th e grow th o f long bone s (Hu ntle y, Bu sh et al. 2003). The gro wth pl ate c ont ain s discr ete tra ns ver se z one s of chondr ocyt e s in differe nt st age s of dif ferenti ation, from th e res ting zone (fart he st from the b one) to t he up per and lo wer prolif erati ng zone s (cl os er to the b one) a nd the h yp ertrophi c zon e (clo se st to t he bone) wh ere c ell s are t ermina lly differ entia ted, larg er, and ar e se cretin g the e xtra cell ular matr ix (EC M) that o st eobl ast s wi ll us e a s

112 a templ ate for bo ne forma tion. Studi e s have show n that p erle can i s es se ntial for lo ng bon e growt h (Arika wa-Hir as awa, W ata nab e et al. 1999; C os tell, Gu st afs son et al. 199 9; Has s ell, Yam ada et al. 200 2) and tha t it is th e maj or HSPG in d evel oping gro wth pl ate c artil age (Govindr aj, We st et al. 2002). Wi thout perle can, gro wth pla te c ell s do not prolif erat e and th e cart ilag e, an d henc e the b one t empl ate, is compromi s ed. Not surpri singl y, perle c an null mi ce ar e fetal d warv es and die b efore or shortl y after birt h (A rikaw a-Hira sa wa, W atan abe et al. 1999 ; Co ste ll, Gus taf ss on et al. 1999). Silverm an-H andm ak er type Dy s se gment al Dy spl a sia (D DSH) and Sch wart z-J amp el Syndrom e (SJS) are t ype s of hum an dw arfism a ls o know n to re sult from a los s or alt erati on of perl ec an (Arikaw a-Hir as aw a, Wilco x et al. 2001; Ari kaw a-Hir as awa and Y ama da 2001 ; Arika wa-H ira saw a, Le et al. 2002). Thi s indi cat es th at per leca n is n ec es sar y for proper ske let al dev elop ment. While t he int erac tion of p erlec an wi th compo nent s foun d in bas eme nt membr ane s i s wid ely do cum e nted, le s s is kn own a bout th e intera ction s of p erlec an wi th the comp onent s in th e growt h plat e. The aim of thi s st udy w as t o ident ify p erlec an– bindi ng prote in s endog eno us to t he grow th pla te an d to chara cter ize t heir int erac tion s with perl ec an. Us ing p erlec an affi nity chroma togra phy, prot ein seq uen cing and immu nohi sto ch emi stry, we ident ified h ist one H 3 a s a bindin g partn er of perl eca n and as a n endog enou s prot ein in th e growth pl ate. U sing a cat ioni c filtrati o n binding as s ay, we furth er chara cter ize d H3’ s inter acti on with p e rleca n as o ne of hig h spe cific ity de pen dent p artly on p erle ca n’s GA G ch ain s.

113 Expe rimenta l Proc edur es

Sourc es of Pe rle can, Antibodi e s and Extra ct. Perlec an w as p urified from w hole f etal bovine rib gro wth pl ate s with 4 M guan idin e hydro chlori de e xtra ction, c esi um chl oride den sity gra die nt ce ntrifug ation, siz e e xclu sion chrom atogr aphy a nd ion exc han ge c hromat ograp hy (Gov ind raj, We st et al. 20 02). Anti- histo ne H3 antib ody w as from U ps tat e Techno logy ( Charlo tte svil le, VA, USA) an d perle ca n anti body (GPP 76) wa s from a comm erci al servi ce (thro ugh Invi troge n Bio tec h) u sing gro wth pl ate p erle can purified in o ur lab. Grow th pla te s wer e also slic ed tra ns ver sely i nto six sequ enti al 1 mm-thi ck sec tion s a s previou sl y de scrib ed (Pla as and San dy 199 3). The sli ce s wer e rins e d in PBS the n the c ell- surfa ce a nd extr ac ellul ar matrix bi otin ylate d for 30 minut es at room temper ature with 0. 5 mg/m l Sulfo NHS LC Biotin (Pi erce Biot echn olog y, Roc kford, IL, USA) in PBS. After blo ckin g the unrea cte d amin es w ith 20 m M gly cine and a fin al rins e in PBS, th e cartil age slic e s were m inc ed an d extra cted for 1 ho ur at 4° C in 5 volume s of 1% Brij- 97 extr acti on buff er (1% Brij 97, 0. 15 M N aC l, 20 mM Tris H Cl, 2 µg/ml l eup eptin, 10 µg/ml aproti nin, 1 mM PMSF). The extr act w as centrif uge d an d the s olubl e mat erial dialy zed agai nst wat er.

Perle can Affinity Ch romato graph y. Purified gro wth pl ate p erlec an w as con jugat ed to NH S-act ivat ed agaro se b ea ds (GE H ealt hcar e, Upp sal a, Swede n) for us e as an affinity c olumn. On e milli gram of perl eca n (or 1 mg of BSA a s control) w as add ed to a 1 ml b ed volu me of wa she d, HCl- acti vat ed

NHS-ag aro se slurry in co upli ng buffer (0. 2 M NaH CO 3 , 0.5 M NaC l). The mixture w a s incu bat ed at r oom temp eratur e for 5 hour s,

114 the liqui d drain ed a nd the u nrea cte d a mine s on the b ea ds bl ock ed with 0. 1 M Tri s HCl o verni ght at 4 °C. The bea ds w ere w as hed seq uent iall y with P BS, PBS/1 M Na Cl and col d wat er. They wer e then w as hed thr ee tim es with al tern ati ng pH 11 (0. 1 M N a 2 C O 3 / 0.5 M Na Cl) an d pH 4 (0. 1 M sodi um ac eta te / 0. 5 M Na Cl) buff ers.

After a wa sh i n 10 mM N a 2 C O 3 and th e n in PBS, the b ead s wer e incub ate d overn ight at 4 °C w ith the 1 % Brij-97 / 0.15 M N a Cl extra ct of cel l-surf ac e biotin ylat ed gro wth plat e c artilag e. Protei ns were el ute d from the co lumn by a non- contin uou s grad ient of N aC l at 0. 15, 0. 5, 1. 0 and 1. 5 M. On e millil i ter fractio ns w ere c olle cte d at ea ch grad ient ste p: half of e ac h frac tion wa s run on S DS-PAGE and blot ted for bio tin or st ain ed wit h Coom as si e. The ban ds e lutin g at 13-19 k Da (p13 /17/1 9) on the Coom as sie- st aine d gel of th e 1.5 M eluat e wer e ex cis ed u sing a razor bl ad e and sent t o the K eck Facilit y (Yal e Univ er sity, Ne w Hav en, CN, USA) for trypti c dige stio n, sep arati on of pe ptid es by r e vers e pha s e chrom atogr aphy, and s equ enc ing of s ele cte d pep tide s.

Glyco sida se Tr eatment of Per lec an. One micr ogram of p erle can pro tein (2 0 0 ng GAG e quiv ale nt) wa s either sha m- dige st ed or dig est ed s ep arately w ith eit her chondr oitin as e AB C (C’ as e, 20 mU) to remove c hondro itin sulfat e GAG s, or with H eparit ina se s I and II ( H’as e, 10 mU e ach) to re mov e hepar an s ulfat e GA Gs. The di ge stion w as in 0. 2 ml of di ge stion buffer (20 mM Tri s-HC l, 50 mM sodi u m acet ate, 5 m M Ca Cl 2 , an d 0. 2 mg/ml BS A, pH 7. 4) for 3 hours at 37°C. All enz yme s are from As soc iat es of Cap e Co d, East Fal mout h, MA.

115 Cationi c Filtration (C AF) As say. Hist one bi ndin g to perl eca n wa s det er mined u sin g the C AF a ss ay a s previou sl y de scrib ed (For sten and Nu g ent 200 1). In brief, Histo ne H3 (H3; R och e Bio ch em, Indian apol is, IN, USA) was l abe led u sin g IODO-G en-co ate d tub es (Pi erce Biot ec h) and s odium io dide (N a 1 2 5 I, Perkin-Elmer, B ost on, M A, USA) by th e est abli sh ed C hiz zonit e method (For st en and Nug ent 200 1). Two nanogr am s of 1 2 5 I-H3 were adde d to CAF b uffer (0. 15 M Na Cl, 50 mM Tris, 2 mg/ml B SA) with 8 ng of growth pl ate p erle can pr otei n (equiv ale nt to 1. 6 ng of glyco sa minog lyc an s [GAG]) a s pre viou sly d es cribe d for determ ining perle can/F GF int eract ion s (Ri sk e, Chiz zonit e et al. 19 90; For sten and Nu gent 2 001; Smit h, We st et a l. 2006) . As com peti tor, chondr oitin sulfa te C or bo vin e kidn ey hepara n su lfate (Si gma, St. Louis, M O, USA) wa s ad ded at con ce n tration s from 0 to 160 n g, that is 0 to 100-f old of the p erle can G AG e quival ent u se d. The rea ction was i ncu bat ed at 30 °C b efore filt ering through Zet a-Prob e membra ne (Bi oR ad, Herc ule s, CA, US A) and co untin g the me mbran e in a TM An alyti c Mo del 11 91 gam ma c ounter. A s con trol s, match ing incub atio ns w ere do ne wit h only 1 2 5 I-H 3 and the 0 t o 160 ng of comp etitor but n o perl eca n. To identif y wheth er the bi ndin g of the radiol abel ed hi sto ne to p erlec an inv olv ed perl eca n’s G AG side chai ns, th e CAF w a s performe d with 1 2 5 I-H3 and s ham-d ige ste d, Chon droitin a se A BC-d ige ste d or Hep ar itina se-d ige ste d perl ec an.

Immunohi stoch emi stry. Frozen tr ans ver se se ction s of wh ole gr owth pl ate c artila ge w ere fixed in a cet one, rin sed i n PBS an d bl ocke d with 1% h eat- inact ivat ed go at s erum (HIGS) a t 37° C humidity. The se ction s w ere incub ate d with bo th mou se anti-H 3 (U pst ate) an d rabbi t anti-

116 perle can (GPP 76) anti bodi es ( eac h dilu ted 1:50 i n PBS) the n with both TRITC- conj ugat ed an ti-mou se an d FITC-conj ugat ed an ti-rabb it antibo die s (Sigm a). The slid e s were m ounte d in aqu eou s mou nting medium (Pi erce Biot ech), co vere d, se a led and t he fluor es ce nt sign al s det ect ed with a micro sc ope.

Statistic s. All mea sur emen ts in t he bin ding a s sa y s are re porte d as t he me an of three or mor e repli cat es ± st and ard err or of the mea n.

Res ults

Perle can Affinity Ch romato graph y. Potenti al perl ec an-bi nding pr otein s in the dev elo ping bo vine ri b growth pl ate w ere i sol ated b y bioti nyl ating th e ti ssu e, extr actin g with mild d eterg ent (Br ij 97), appl ying the extra ct to a p erle can affinity c olumn and el uting t he bou nd protein s wit h a st ep gra die nt of incre asi ng Na Cl. Figur e 19 s how s a n anti-bio tin W est ern blo t of sel ect ed frac tion s elu ted from a p erle c an colu mn (+) an d a BSA control colum n (-). The band s at 13, 1 7 and 19 k Da th at elut ed at high s alt co nc entrat ion s wer e con sid er ed stro ng bin ding p artner s of perle can. The l ane l abel ed “ *” s how s a Cooma s sie b lue stai n of a 1. 5 M s alt fract ion from th e perle ca n a ffinity col umn. The ba nd s label ed p1 3, p17 and p 19 wer e ex ci sed from the gel a nd chara cter ize d by s equ enc ing. This id en tified p1 3 as h ist one H 4, p17 as hi sto ne H2 A, and p1 9 a s his tone s H 2B an d H3 (Ke ck fa cility, Yale U niv ersit y, Figure 19).

117 Cationi c Filtration A s say. The ins et in Figur e 20 s how s th at the r etent ion of radi olab ele d Hist one H 3 on the cati onic Zet a-prob e filter wa s s ignifi cant ly higher in th e pre se nce of p erle can (bl a ck bar) com pare d to it s retenti on in th e ab sen ce of p erle can (w hite bar). The ma in figure sho ws th at thi s int erac tion w as n ot yet 50% inhibit ed ev en wit h the additi on of 100-fol d ex ce ss of c omp eti ng chon droitin sulf ate a nd hepar an s ulfat e (CS an d HS). Both G A Gs prod uc ed si milar inhibit ory effe cts o n the bi nding of H 3 to perlec an. The e nzym ati c remova l of chon droitin sulf ate G AG s from the cor e protei n of perle can re duc ed it s bindi ng to rad iola bele d hist one H3 by 17% where a s remov al of the h epar an s ulfat e chain s red uce d the bi nding by 38% (Figure 2 1).

Immunohi stoch emi stry. The loca tion of bo th perl ec an and h ist one H3 in a repre se ntati ve trans ver se sec tion of th e growt h plat e is s hown as d etermi ned b y stai ning w ith ant ibodi e s to perl ec an an d H3 (Figure 22). Perl ec an i s local ize d to the t erritoria l (or peric ell ular) matrix around t he c ell. Hist one H 3 al so loc ali ze s to thi s are a i n the se int act (ie. no n- perme abili zed) c ell s. There ar e are as, howe ver (*) wher e ea ch protein i s pre se nt s epar atel y from the other. The da sh ed re ctan gle sho ws a t ypic al ch ondro cyt e (cle ar reg ion) with it s s urroundi ng territoria l/peri cel lular m atrix dire ctly around th e c ell. In the right pane ls, th e neg ativ e contr ols ( stai ned only with FIT C-con juga ted or TRITC-con jug ated se cond ary an tibod y and no prim ary ant ibod y) sho wed no b ac kgroun d st ainin g of the se ction s.

118 Dis cus sio n

The res ults of o ur stu dy indi cat e tha t h iston e H3 i s pre se nt in the peri cell ular ma trix of the chon droc yte s in the d evel opin g growth plate, wh ere it c olo cali ze s with t he pro teogly ca n perle ca n. Chrom atogr aphy of a mi ld Brij-9 7 extr act of de velo ping gro wth plate cartil age o ver a p erle can affi nity column (Fig ure 19) s how ed the cor e his tone s H 2A, H2 B, H3 and H 4 as p erlec an-bi ndin g protein s. Previ ou s sc ientif ic s tudi es h a ve sh own th at hi ston e s, both free and compl exe d into nu cle os ome s, can bin d to he parin a nd to hepar an s ulfat e (Schmi ede ke, Sto ckl et al. 1989; Bi lozur and B is wa s 1990; W at son, Goo derh am et al. 19 99; Henriq uez, Ca sar et al. 2002). Wat so n et al (W at son, Goo derh am et al. 199 9) confirm ed th at histo ne s wer e pre sen t on the p la sma m embran e of ac tivat ed T- lympho cyt es. Thi s pre sen ce of nu cle os ome s on the T-c ell pla sm a membra ne de cre as ed by 90 % if the cel ls wer e dig est ed wit h hepari na se or h eparit ina se - en zym es t hat cl eav e, resp ect ivel y, region s of high and lo w sulf atio n in he paran sulfa te. They conc lude d that h epar an sulfat e prot eog lyca ns k ept th e his tone s bound to th e c ell s urfac e. Schmi ede ke et al (Schm ied eke, Sto ckl et al. 1989) al so sho wed bi nding of hi st o nes t o hep aran sulfa te. In their work, th e hep aran sulf ate w as a p art of the glom erul ar bas eme nt membr ane of t he kid ney, th e organ from whi ch p erle can was i dent ified as a m ajor HSP G. They even pr opo sed t hat th e histo ne s pre se nt in nu cleo som e s circu l ating in th e kid ney c ould a t som e point swit ch th eir nucl eo som al D NA partn er for the gl omerul ar HSPG, to whi ch the y boun d more a vidl y than to D NA. Al so, rese arch er s (Bilo zur an d Bi sw as 19 90) have p artiall y purifie d and chara cter ize d sp ecifi c hep aran sulfa te binding pro tein s in a lu ng carci noma cell li ne an d ide ntifie d the m as hi ston e s.

119 The bindin g of his ton es to h epar an sul fate wo uld be of lit tle phys iolog ica l sig nific anc e in ti ss ue s if the his tone s w ere co nfine d to the ce ll nuc leu s. How ever, hi sto ne s ha ve be en found i n extra cel lular matrix, in cyt opl asm, a nd on pl asm a m embran e of non- apopt otic cell s (M ech eri, Dan nec ker et al. 1993; Wat son, Edw ards et al. 1995 ; Henriq uez, Ca sar et al. 2002). He nriqu ez et a l hav e sh own hi sto ne H1 to be in ske let al mu scl e cell extra c ellul ar matrix, wh ere it colo cali ze s with p erle can (H enriq ue z, Ca sar et al. 2002). M ech eri et al found th at th e nucl eo som al core h ist one H2 B wa s e xpre s sed strong ly on th e surf ace of B ce lls i sol a ted from hum an peri pher al blood (M ec heri, Da nne cker e t al. 1993 ). Watso n et al h ave als o docum ent ed the pr es en ce of hi ston es o n T-cell s urfac e (Wat so n, Edward s et al. 19 95). Thes e prev iou s resu lts h elp to supp ort our finding s tha t the hi sto ne s ca n exi st ex t racell ularl y in cho ndroc yte s. The labe ling of th e hi ston es b y the c el l-imperme abl e bioti nyla tion reage nt Sulfo N HS-LC-bio tin (Figur e 1 9), their extra ctio n from cartil age w ith the m ild Brij- 97 det erge nt and th e immunoh ist och emi stry of the extra cel l ular matrix, in dic ate s tha t the histo ne s are i n the gro wth pl ate c hondr ocyt e matrix, in th e s ame region a s p erlec an. The inter actio n of perl eca n with hi sto n e H3 wa s ch ara cteri zed usin g the c atio nic filtr ation a s sa y (Fig ures 2 0 and 21). The bi ndin g curve s s how th e int eract ion b etwe en H 3 and perl ec an: 50% inhibiti on of thi s bind ing w as n ot rea c hed ev en wit h addi tion of 100-fold c ompe ting c hondr oitin sulfat e and hep aran sulf ate (Figur e 20). Histo ne H3 h a s bee n sh own to b in d hepar an s ulfat e in lung tis sue ( Bilo zur and Bi swa s 19 90). How ever, in our a s say, it see ms to be bindi ng perl ec an’ s chon droiti n sulf ate GA G s as well. The CAF as sa y al so sh ow s tha t there re main s so me bindi ng tha t can not be inhibit ed co mplet ely b y comp etin g GA Gs an d that r emov al of

120 chondr oitin sulfa te from perl ec an red u ced it s bind ing to radiol abel ed hi sto ne H3 b y 17% and re moval of th e hep aran sulf ate chai ns re duc ed the b indin g by 38% but did not elimi nat e bind ing (Figure 21). This sugg es ts th at th e cor e protei n of perle can i s contrib uting to t he bin ding of hi sto ne H3. The immuno st ainin g of the gro wth pl at e sho ws p artia l overl ay of histo ne H3 and p erle can (Fig 2 2) givin g more su pport to th e in vitro intera ction se en b etwe en th e two. Ther e is no sign al for the pro tein s on the cho ndro cyte it s elf (the cl ear re gion in the da sh ed rect angl e) but stai ning i s ap pare nt in the m atrix imm ediat ely surroun ding th e c ell (territorial or p eric ellul ar matrix) a nd in the int erterritori al ma trix betw een th e ce lls. Ther e is b oth more i nten se a nd more w idel y dis tribut ed stai ning for p erlec an th an for hi ston e H3, whic h is e xpe cted sin ce perle can i s kno wn to b e the ma jor HSP G in the ex trac ellul ar matri x of the growth pl ate (G ovindr aj, We st et al. 2002) and i s co nsi dere d to be a struc tural c ompo nent as w ell. Not e tha t des pite th e ext en siv e colo cali zati on of the t wo prot ein s, ther e are s till are a s wher e only perle can (gr een) or H3 (re d) fluore sc e nce i s det ect ed. A number of studi e s hav e sh own th e bi nding of hi ston e s to hep aran sulfa te an d perl eca n (Schmi ede ke, Sto ckl et al. 19 89; B iloz ur and Bis wa s 1990; W at son, Goo derh am et al. 19 99; Henriq uez, Ca sar et al. 2002). Howe ver, our ide ntific atio n of his tone H3 in the d eve lopin g growt h plat e cartil age a nd th e invol vem ent of the G AG ch ain s and th e cor e prote in of perle can in H3 bind ing ar e nove l findi ngs. Furth ermore, th e dis cov ery of the hi ston e’s pro ximity t o perl eca n in growth pl ate c artil age sup ports t he pos sibi lity of in vi vo inter act ion s bet w een th e two. The se d ata hi ghlig ht new po s sibili tie s for de scri bing th e fu nction of p erle can i n cell proliferat ion no t only thro ugh gro wth f actor s an d cell- sig nali ng mol ecul e s (Iozzo, Co hen et al. 199 4; Mo ngia t, Taylor et al. 200 0; Gov indra j, We st et al. 2006; Smit h, We st et a l. 2006) but als o via el eme nt s of the

121 trans cripti onal m ach inery it s elf. This i ntera ction could allow p erle ca n to exert a dir ect l eve l of contro l on cho n drocyt e act ivity, a s ha s pre viou sly been sho wn for perl eca n and h ist one H 1 in regen erati ng mu scl e ce lls (Henriqu ez, C a sar et a l. 2002). Intere stin gly, in add ition to o ur dat a s howing h ist one H 3’s co- occurr enc e and i ntera ctio n with p erle c an in the gr owth pl ate, th e chroma tin a s soci atin g protei ns hi st one deac etyl as e (HD AC)-3 a nd HD AC4 are al so kn own to aff ect c hondr ocyt e a ctivity and lo ng bon e growt h. HDA C3, whi ch remo ve s ac etyl gro up s from the cor e his tone s to c au se gene re pre ss ion, is k nown t o regul ate ost eobl ast d ifferen tiati on an d bon e formation b y inhib iting th e o steo cal cin gene vi a the c hondr ocyt e-s pe cific runx2 tran scr iptio n factor (Sc hroe der, Kahl er et al. 200 4). Ost eobl as ts ar e the ce lls t hat rep lac e the m iner aliz ed c artilag e matri x produ ced b y growth plate chon drocy te s with a coll age n-ric h matrix, lea ding to t he forma tion of new bon e. HDA C4 is al so kno wn to co ntrol bo ne formation v ia runx 2 by inhibiti ng the h ypertr ophi c cho ndroc yt e phen otyp e, allow ing prol iferati ng chondr ocyt e s to avoi d prem ature h yper trophy an d thu s prem ature cartil age os sific ation (V eg a, Mat su da et al. 200 4). One mec hani sm b y whic h HDA C4 do es th is i s by suppr es sin g ac e tylatio n of his ton e H3 on th e runx2 promoter. Wit h HDA C4 abs ent in a mo use k noc kout mo del, ther e is n o dea cety latio n of H3 on th e runx2 pr om oter and t he runx 2 gen e be come s activ e, lea ding to pr emat ure hyp ertrop hy of chon drocy te s and, thu s, premat ure o ssifi cat ion of s kel etal elem ent s. Bec au se of thi s, th e HDA C4 null mic e suff er from fetal d warfi sm, d ecre as ed mob ility du e to bon e malformat ion a nd an in abili ty to bre at he due to t hora cic d eformiti es, chara cter isti c s all see n in the p erle can knocko ut mou se mo del. Perh ap s the int eract ion s bet we en perl ec an an d histo ne H3 al so invo lve H DA C4 an d runx2 in co ntrolli ng a spe ct s of the c o mplex m ech ani sm of long b one growth.

122 Ackno wledgm ent

This work w a s su pport ed by gra nts fro m the Shriner s Ho spi tal s for Childr en, North A meric a (to J RH).

123 Figure s Figure 19.

Affinity purifi cat ion of perl ec an’ s bin ding part ner s. Anti-bi otin w est ern blot of s ele cted fra ctio ns from a mi ld deterg ent e xtra ct inc ubat ed ov er a perle can affi nity c olumn (+) or a contr ol, BSA-co upl ed col umn (-) are sho wn. The 0. 15 M wa she s an d s ele cte d salt elua te s at 0. 5 M and 1. 0 M are sh own. The b and s for p13, p17 and p19 are ind ica ted in th e lan e label ed “ *” s howi ng a C ooma s sie sta in of a 1.5 M elu ate fra ction. The p13/17 /19 ba nd s were i ndivi dual ly ex c ise d and sent t o the K eck fa cilit y for sequ en cing.

124 Figure 20.

Cati onic Fil tratio n (CAF) A s say s show that perl eca n bind s to 1 2 5 I-hi ston e H3. The bindi ng of H3 wit hout (-) and with (+) 8 ng of perl ec an i s sh own in CPM i n the in se t. Specifi c bin ding i s the diff eren ce b etwe en bi nding of H3 to the Zet a-Prob e membr ane w ith p erlec an in th e rea ction mi nu s the nons pe cific bi nding t o the Zet a-Probe alon e. The sp ecifi c bindi ng to perle can i s ca lcul ate d and sho wn a s % of control in t he pre se nc e of comp etitor s in the m ain figur e. Unla be led com petit ors w ere e ither chondr oitin sulfa te C ( CS) or bovin e k idney h epar an s ulfat e (HS).

125 Figure 21.

Effect of chon droiti na se a nd hep aritin as e pre-tre atme nt on th e bindi ng of perle can to 1 2 5 I-hi ston e H3. Eight n ano grams of p erle can w a s first dige ste d with Chon droitin as e A BC or with He pariti na se I an d II for 3 hours at 37° C or wa s left un dig est ed b efore be ing u sed i n the C AF a ss ay s.

126 Figure 22.

Immunolo cali zat ion of Hi sto ne H3 and Perlec an in th e dev elopi ng grow th plate. Immun ohi stoc hemi stry of gro wt h plate se ction s show s th e pre sen ce of perle can (gre en) an d hi ston e H3 (re d). The merged im age sho ws t he colo cali zati on of the t wo prot ein s (yell ow/ora nge c olor) an d se lec ted area s wh ere th e two ar e se par ate (*). A repre sen tativ e ch ondro cyt e is outlin ed with a da she d rect angl e in e ac h panel. In th e top p anel, th e cle ar chondr ocyt e ce ll body and th e st ain ed peric ellul ar/territ orial m atrix around th e c ell are sho wn. Scal e bar s = 10 micro n. The right pa nel s s how the con trol s taini ng wit h only l abel ed se cond ary ant ibod y but no prim ary.

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130

CHAPT ER THR EE

CONC LUS IONS

T h e g r o w t h p l a t e i s t h e p r i m a r y r e g u l a t o r o f e n d o c h o n d r a l b o n e g r o w t h i n h u m a n s a n d a n i m a l s . E n d o c h o n d r a l b o n e s a r e f o r m e d f r o m a c a r t i l a g e i n t e r m e d i a t e t h a t i s s e c r e t e d b y c e l l s i n t h e g r o w t h p l a t e ( c h o n d r o c y t e s ) a n d u s e d a s a t e m p l a t e f o r b o n e p r o d u c t i o n ( P i n e s a n d H u r w i t z 1 9 9 1 ) . T h e f o r m a t i o n o f c h o n d r o c y t e s f r o m m e s e n c h y m a l s t e m c e l l s ( M S C s ) i s a c o o r d i n a t e d p r o c e s s , r e g u l a t e d m a i n l y b y t h e t r a n s c r i p t i o n f a c t o r S o x 9 ( H u a n g , C h u n g e t a l . 2 0 0 1 ) . A l t h o u g h t h e g r o w t h p l a t e h a s o n l y a s i n g l e c e l l t y p e , t h e c h o n d r o c y t e , t h e r e a r e t h r e e t r a n s v e r s e z o n e s o f c h o n d r o c y t e s a t d i f f e r e n t s t a g e s o f a c t i v i t y a n d m a t u r i t y , r a n g i n g f r o m t h e q u i e s c e n t s t e m - l i k e ‘ r e s t i n g ’ c h o n d r o c y t e s n e a r e s t t h e e p i p h y s e a l b o n e , t o t h e a c t i v e l y r e p l i c a t i n g ‘ p r o l i f e r a t i n g ’ c h o n d r o c y t e s a n d t h e l a r g e r , l e s s a c t i v e ‘ h y p e r t r o p h i c ’ c h o n d r o c y t e s . T h i s l a s t z o n e o f c e l l s s e c r e t e s t h e m a t r i x t h a t b o n e - f o r m i n g c e l l s ( o s t e o b l a s t s ) r e m o d e l i n t o n e w b o n e ( F a r n u m a n d W i l s m a n 1 9 8 9 ; M i n i n a , K r e s c h e l e t a l . 2 0 0 2 ) . T h e a c t i v i t y o f t h e c h o n d r o c y t e s i n t h e g r o w t h p l a t e i s a l s o t i g h t l y c o n t r o l l e d , w i t h m u l t i p l e e n v i r o n m e n t a l , g e n e t i c , h o r m o n a l a n d e v e n n u t r i t i o n a l f a c t o r s a f f e c t i n g t h e a c t i v i t y o f t h e s e c e l l s . P r o t e o g l y c a n s p l a y i m p o r t a n t r o l e s i n g r o w t h p l a t e c a r t i l a g e s t r u c t u r e a n d f u n c t i o n . A g g r e c a n i s t h e m a j o r

131 p r o t e o g l y c a n i n c a r t i l a g e . I t c o n t a i n s o v e r 1 0 0 c h o n d r o i t i n s u l f a t e ( C S ) c h a i n s t h a t b i n d w a t e r a n d s e r v e t o p r o v i d e t h e “ v o l u m e ” o f t h e g r o w t h p l a t e e x t r a c e l l u l a r m a t r i x ( E C M ) . N a t u r a l a g g r e c a n k n o c k o u t s ( R i t t e n h o u s e , D u n n e t a l . 1 9 7 8 ; L i , S c h w a r t z e t a l . 1 9 9 3 ; W a t a n a b e , K i m a t a e t a l . 1 9 9 4 ) h a v e s h o r t l i m b s d u e t o t h e r e d u c e d s i z e o f t h e E C M i n t h e g r o w t h p l a t e . P r o t e o g l y c a n s a r e a l s o e s s e n t i a l i n t h e i r b i n d i n g o f g r o w t h f a c t o r s i n c a r t i l a g e , p r o t e c t i n g t h e g r o w t h f a c t o r s f r o m d e g r a d a t i o n a n d a l s o s e r v i n g t o a l t e r n a t e l y s e q u e s t e r g r o w t h f a c t o r s f r o m o r d e l i v e r t h e m t o t h e i r g r o w t h f a c t o r r e c e p t o r s ( K n u d s o n a n d K n u d s o n 2 0 0 1 ) . P e r l e c a n i s t h e p r i m a r y h e p a r a n s u l f a t e p r o t e o g l y c a n ( H S P G ) i n g r o w t h p l a t e c a r t i l a g e ( G o v i n d r a j , W e s t e t a l . 2 0 0 2 ) . M o u s e m o d e l s h a v e p r o v e n t h a t p e r l e c a n i s e s s e n t i a l f o r g r o w t h p l a t e s t r u c t u r e a n d f o r p r o p e r e n d o c h o n d r a l b o n e g r o w t h ( A r i k a w a - H i r a s a w a , W a t a n a b e e t a l . 1 9 9 9 ; C o s t e l l , G u s t a f s s o n e t a l . 1 9 9 9 ) . T h e m a j o r e f f e c t o f p e r l e c a n i n l o n g i t u d i n a l b o n e g r o w t h i s o n p r o l i f e r a t i o n . P e r l e c a n a f f e c t s g r o w t h p l a t e c h o n d r o c y t e p r o l i f e r a t i o n v i a t h e g r o w t h f a c t o r s t h a t t h i s m u l t i - d o m a i n p r o t e o g l y c a n b i n d s n o t o n l y o n t h e v a r i o u s r e g i o n s o f i t s c o r e p r o t e i n b u t a l s o o n t h e g l y c o s a m i n o g l y c a n ( G A G ) c h a i n s a t t a c h e d t o i t s c o r e p r o t e i n ( I o z z o , C o h e n e t a l . 1 9 9 4 ) . T h e t y p e , s i z e a n d s e q u e n c e o f t h e G A G s o n p e r l e c a n v a r y d e p e n d i n g o n t h e t i s s u e o r i g i n . I n g r o w t h p l a t e c a r t i l a g e , p e r l e c a n h a s c h o n d r o i t i n s u l f a t e ( C S ) c h a i n s o f 3 7 - 4 2 k D a a n d h e p a r a n s u l f a t e c h a i n s o f 2 0 - 2 2 k D a ( W e s t , G o v i n d r a j e t a l . 2 0 0 6 ) . T h e C S c h a i n s a c c o u n t f o r 7 5 % o f t h e t o t a l G A G o n p e r l e c a n a n d t h e H S a c c o u n t s f o r t h e o t h e r 2 5 % .

132 P a p e r I W e p r e v i o u s l y k n e w t h a t p e r l e c a n H S c o u l d b i n d F G F - 2 b u t c o u l d n o t d e l i v e r i t t o i t s g r o w t h p l a t e F G F R ( G o v i n d r a j , W e s t e t a l . 2 0 0 6 ) . F G F - 2 i s a p l e i o t r o p i c g r o w t h f a c t o r t h a t i s p r e s e n t i n c a r t i l a g e a n d n e g a t i v e l y r e g u l a t e s g r o w t h p l a t e c h o n d r o c y t e p r o l i f e r a t i o n , s i n c e m i c e o v e r - e x p r e s s i n g F G F - 2 a r e s k e l e t a l d w a r v e s . I n P a p e r I o f t h i s d i s s e r t a t i o n ( S m i t h , W e s t e t a l . 2 0 0 6 ) , w e u s e d t h e c a t i o n i c f i l t r a t i o n ( C A F ) a n d i m m u n o p r e c i p i t a t i o n ( I P ) b i n d i n g a s s a y s t o s h o w t h a t p e r l e c a n p u r i f i e d f r o m t h e d e v e l o p i n g g r o w t h p l a t e b o u n d t o F G F - 2 i n d e p e n d e n t l y o f a n y o t h e r g r o w t h p l a t e m o l e c u l e s ( F i g u r e 5 ) . W e f o u n d t h a t t h e F G F - 2 b i n d i n g w a s p r i m a r i l y t o t h e H S c h a i n s o f p e r l e c a n ( F i g u r e s 6 a n d 7 ) . D i g e s t i o n o f t h e H S c h a i n s s i g n i f i c a n t l y r e d u c e d F G F - 2 b i n d i n g t o p e r l e c a n ( F i g u r e 6 ) a n d b o v i n e k i d n e y ( b k ) H S a s a c o m p e t i t o r r e d u c e d p e r l e c a n : F G F - 2 b i n d i n g b y 9 0 % ( F i g u r e 7 ) . W e c o n c l u d e d , t h e n , t h a t t h e H S c h a i n s o f p e r l e c a n a r e t h e e s s e n t i a l p l a y e r s i n t h e m a x i m a l b i n d i n g o f F G F - 2 t o p e r l e c a n . W e a l s o s h o w e d t h a t h e p a r i n c o u l d e n h a n c e t h e b i n d i n g o f F G F - 2 t o i t s c e l l - s u r f a c e F G F R , c o n f i r m i n g a p r e v i o u s l y s h o w n r e l a t i o n s h i p ( O r n i t z , Y a y o n e t a l . 1 9 9 2 ) . W e c o n c l u d e d f r o m t h e s e e x p e r i m e n t s ( F i g u r e s 1 0 a n d 1 1 ) t h a t p e r l e c a n c o u l d b i n d F G F - 2 b u t c o u l d n o t d e l i v e r i t t o F G F R - 1 a n d - 3 u n l e s s t h e C S c h a i n s w e r e r e m o v e d . F G F R - 1 a n d - 3 a r e t h e g r o w t h p l a t e F G F R s a n d a r e d i r e c t r e g u l a t o r s o f g r o w t h p l a t e c h o n d r o c y t e p r o l i f e r a t i o n ( S h i a n g , T h o m p s o n e t a l . 1 9 9 4 ; M u e n k e a n d S c h e l l 1 9 9 5 ; W h i t e , C a b r a l e t a l . 2 0 0 5 ) . W e t h u s c o n c l u d e d t h a t t h e C S c h a i n s o n d o m a i n s I a n d V o f g r o w t h p l a t e p e r l e c a n ( i e . t h e N - a n d C - t e r m i n a l e n d s ) p r e v e n t d e l i v e r y o f t h e H S - b o u n d F G F - 2 t o i t s F G F R . W e s p e c u l a t e t h a t t h i s i s d u e t o t h e l a r g e r s i z e o f t h e C S c h a i n s w h i c h c o u l d s t e r i c a l l y h i n d e r t h e

133 t r a n s f e r o f t h e H S - b o u n d F G F - 2 t o F G F R - 1 a n d - 3 . T h e s u b s t i t u t i o n o f g r o w t h p l a t e p e r l e c a n w i t h C S i s t h u s a r e g u l a t o r o f t h e d e l i v e r y v e r s u s s e q u e s t r a t i o n a c t i v i t i e s o f p e r l e c a n i n t h e g r o w t h p l a t e a n d t h u s c o n t r o l s t h e a c t i o n o f g r o w t h f a c t o r s o n t h e c h o n d r o c y t e s . T h e F G F - 2 : F G F R - 3 m e d i a t e d p a t h w a y o f G P c h o n d r o c y t e i n h i b i t i o n i n v o l v e s I n d i a n h e d g e h o g ( I h h ) ; w h e n F G F R - 3 i s b o u n d b y g r o w t h f a c t o r s , i t i n h i b i t s I h h e x p r e s s i o n a n d p r e v e n t s c h o n d r o c y t e p r o l i f e r a t i o n ( M i n i n a , K r e s c h e l e t a l . 2 0 0 2 ) . I t w o u l d b e i n t e r e s t i n g t o s e e t h e d y n a m i c s o f t h i s F G F - 2 : F G F R - 3 : I h h : p e r l e c a n r e l a t i o n s h i p . W e k n o w t h a t p e r l e c a n H S c a n b i n d F G F - 2 a n d i s a n e c e s s a r y c o f a c t o r i n t h e s u b s e q u e n t b i n d i n g o f F G F - 2 t o F G F R - 3 ( O r n i t z 2 0 0 0 ; O r n i t z a n d M a r i e 2 0 0 2 ) . W e a l s o k n o w t h a t p e r l e c a n h a s a n L D L - r e c e p t o r - l i k e r e g i o n i n i t s d o m a i n I I ( N o o n a n , F u l l e e t a l . 1 9 9 1 ) . D o m a i n I I c o u l d b i n d t h e c h o l e s t e r o l m o i e t y k n o w n t o b e o n I h h ( S t - J a c q u e s , H a m m e r s c h m i d t e t a l . 1 9 9 9 ) . W e c o u l d i n v e s t i g a t e ( u s i n g t h e C A F o r I P b i n d i n g a s s a y s ) w h e t h e r p e r l e c a n b i n d s I h h . P e r l e c a n : I h h b i n d i n g w o u l d a d d a n o t h e r l e v e l o f c o n t r o l t o t h i s r e g u l a t i o n o f c h o n d r o c y t e a c t i v i t y . T h e d o c u m e n t e d c h a n g e s i n p e r l e c a n f r o m o n e z o n e o f t h e g r o w t h p l a t e t o t h e n e x t i n c l u d e c h a n g e s i n t h e C S c h a i n s i z e a n d s u l f a t i o n p a t t e r n ( W e s t , G o v i n d r a j e t a l . 2 0 0 6 ) . T h e r e f o r e , i n t h e h y p e r t r o p h i c a n d l o w e r p r o l i f e r a t i n g z o n e , w h e r e p e r l e c a n h a s l a r g e r C S c h a i n s , F G F - 2 w o u l d b e s e q u e s t e r e d a w a y f r o m t h e r e c e p t o r m o r e t h a n i t w o u l d b e i n t h e u p p e r p r o l i f e r a t i n g a n d t h e r e s t i n g z o n e s , w h e r e p e r l e c a n h a s l e s s C S , h e n c e t h e d i f f e r e n t p r o l i f e r a t i v e a c t i v i t i e s o f c e l l s i n t h e s e z o n e s .

134 P a p e r I I I t w a s p r e v i o u s l y k n o w n t h a t F G F - 1 8 b i n d s t o h e p a r i n a n d a l s o t o H S . H o w e v e r , w e a r e t h e f i r s t t o s h o w t h a t F G F - 1 8 a l s o b i n d s t o g r o w t h p l a t e p e r l e c a n ( P a p e r I I ) . F G F - 1 8 i s e x p r e s s e d i n t h e p e r i c h o n d r i a l l a y e r s u r r o u n d i n g t h e g r o w t h p l a t e , m a k i n g i t a c a n d i d a t e r e g u l a t o r o f g r o w t h p l a t e f u n c t i o n . F G F - 1 8 i s e s s e n t i a l f o r p r o l i f e r a t i o n o f g r o w t h p l a t e c h o n d r o c y t e s a n d f o r d i f f e r e n t i a t i o n o f t h e p r o l i f e r a t i n g c h o n d r o c y t e i n t o h y p e r t r o p h i c c h o n d r o c y t e s . I t a l s o r e g u l a t e s o s t e o g e n e s i s a n d v a s c u l o g e n e s i s i n g r o w t h p l a t e c a r t i l a g e . N o t s u r p r i s i n g l y , t h e n , F G F - 1 8 n u l l m i c e h a v e s e v e r e a b n o r m a l i t i e s i n l o n g b o n e g r o w t h ( L i u , X u e t a l . 2 0 0 2 ; O h b a y a s h i , S h i b a y a m a e t a l . 2 0 0 2 ; L i u , L a v i n e e t a l . 2 0 0 6 ) . F G F - 1 8 a f f e c t s c h o n d r o c y t e s t h r o u g h F G F R - 3 ( D a v i d s o n , B l a n c e t a l . 2 0 0 5 ) , o s t e o g e n e s i s t h r o u g h F G F R - 1 a n d / o r - 2 ( L i u , X u e t a l . 2 0 0 2 ) a n d v a s c u l o g e n e s i s t h r o u g h V E G F ( G e r b e r , V u e t a l . 1 9 9 9 ) . W e r e p o r t t h a t F G F - 1 8 b i n d s t o p e r l e c a n v i a d o m a i n I I I o f p e r l e c a n ’ s c o r e p r o t e i n . D o m a i n I I I h a s a r e p e t i t i v e s t r u c t u r e a n d h a s b e e n s h o w n t o h a v e m u l t i p l e b i n d i n g p a r t n e r s . B y b i n d i n g h e r e , F G F - 1 8 i s p o s s i b l y b e t t e r p o s i t i o n e d t o i n t e r a c t w i t h m u l t i p l e g r o w t h f a c t o r s a n d r e c e p t o r s i n c a r t i l a g e . B i n d i n g t o p e r l e c a n w o u l d g i v e t h e p e r i c h o n d r i a l l y e x p r e s s e d g r o w t h f a c t o r a n a c t i v e l i n k t o t h e g r o w t h p l a t e c a r t i l a g e a n d t o i t s F G F R o n t h e c h o n d r o c y t e s u r f a c e . W e d o n o t k n o w y e t w h i c h r e g i o n o f d o m a i n I I I ( s u b d o m a i n s I I I a , I I I b o r I I I c ) a n d , f u r t h e r , w h i c h r e g i o n o f t h e s e s u b d o m a i n s ( c y s t e i n e r i c h r o d - l i k e o r c y s t e i n e - f r e e g l o b u l a r ) r e g i o n s F G F - 1 8 b i n d s . T o i n v e s t i g a t e t h i s , w e c o u l d d i g e s t t h e r e c o m b i n a n t d o m a i n I I I p r o d u c t ( t o w h i c h F G F - 1 8 b o u n d i n o u r e x p e r i m e n t s ) w i t h V - 8 p r o t e a s e . T h i s e n z y m e d i g e s t s w h o l e p e r l e c a n o r d o m a i n I I I t o p r o d u c e a 4 4 k D a a n d a

135 4 6 k D a p r o t e a s e - r e s i s t a n t d o u b l e t o f f r a g m e n t s ( L e d b e t t e r , F i s h e r e t a l . 1 9 8 7 ; C h a k r a v a r t i , H o r c h a r e t a l . 1 9 9 5 ) . T h e 4 4 k D a f r a g m e n t i s d e r i v e d f r o m d o m a i n I I I a a n d t h e 4 6 k D a f r a g m e n t i s f r o m d o m a i n I I I b . D i g e s t i n g t h e w h o l e p e r l e c a n o r d o m a i n I I I w i t h V 8 - p r o t e a s e a n d u s i n g F G F - 1 8 i n a n o v e r l a y a s s a y ( u s i n g a u t o r a d i o g r a p h y t o s e e b o u n d 1 2 5 I - F G F - 1 8 o r W e s t e r n b l o t t o s e e u n l a b e l l e d F G F - 1 8 ) w o u l d s h o w i f t h e g r o w t h f a c t o r b o u n d t o d o m a i n I I I a o r I I I b ) . I f n e i t h e r o f t h e s e s u b d o m a i n s i s t h e b i n d i n g r e g i o n f o r F G F - 1 8 , m o r e s t r i n g e n t d i g e s t i o n , e g s . w i t h h e p a r i t i n a s e , p l a s m i n , s t r o m e l y s i n o r c o l l a g e n a s e ( M o n g i a t , T a y l o r e t a l . 2 0 0 0 ) c o u l d l i b e r a t e a d d i t i o n a l r e g i o n s o f d o m a i n I I I w h i c h c o u l d t h e n b e i d e n t i f i e d w h e n t h e d i g e s t s a r e u s e d i n o v e r l a y a s s a y s .

P a p e r I I I W e h a v e s h o w n i n P a p e r s I a n d I I t h a t p e r l e c a n b i n d s F G F - 2 a n d F G F - 1 8 , m o s t l i k e l y t o s t o r e t h e s e e s s e n t i a l m o l e c u l e s i n t h e d e v e l o p i n g g r o w g r o w t h p l a t e s o t h a t t h e y a r e p r o t e c t e d b u t a v a i l a b l e w h e n n e e d e d . H i s t o n e s , l i k e g r o w t h f a c t o r s , a r e a l s o d e v e l o p m e n t a l l y i m p o r t a n t p r o t e i n s . H i s t o n e s a n d D N A f o r m n u c l e o s o m e s t h a t s e r v e t o c o m p l e x m a m m a l i a n g e n e s i n t o i n a c t i v e h e t e r o c h r o m a t i n . U p o n m o d i f i c a t i o n ( v i a a c e t y l a t i o n o r d e m e t h y l a t i o n ) t h e h i s t o n e s ’ a f f i n i t y f o r D N A i s d e c r e a s e d d u e t o t h e i r n o w m o r e n e g a t i v e c h a r g e , t h e y a r e r e p e l l e d f r o m t h e D N A a n d l e a v e t h e D N A a c c e s s i b l e t o t r a n s c r i p t i o n f a c t o r s a n d o t h e r e l e m e n t s o f t h e t r a n s c r i p t i o n a l m a c h i n e r y , w h i c h c a u s e s g e n e t r a n s c r i p t i o n ( S c h m i e d e k e , S t o c k l e t a l . 1 9 8 9 ) . W e r e p o r t i n P a p e r I I I o f t h i s d i s s e r t a t i o n t h a t p e r l e c a n b i n d s t o H 3 b y b o t h i t s c o r e p r o t e i n a n d i t s G A G c h a i n s . H i s t o n e H 3 i s , l i k e t h e o t h e r h i s t o n e s , n o r m a l l y e x p r e s s e d

136 n u c l e a r l y . B u t h i s t o n e s h a v e a l s o b e e n i d e n t i f i e d e x t r a - n u c l e a r l y a n d e v e n e x t r a c e l l u l a r l y ( W a t s o n , E d w a r d s e t a l . 1 9 9 5 ; H e n r i q u e z , C a s a r e t a l . 2 0 0 2 ) . I n o u r w o r k , w e s h o w t h a t H 3 i s a p a r t o f t h e g r o w t h p l a t e c a r t i l a g e e x t r a c e l l u l a r m a t r i x ( E C M ) w h e r e i t c o l o c a l i z e s w i t h p e r l e c a n i n t h e p e r i c e l l u l a r m a t r i x s u r r o u n d i n g t h e c h o n d r o c y t e s ( F i g u r e 2 2 ) . W e p r o p o s e t h a t , h e r e , a p o o l o f H 3 c a n b e s t o r e d b y p e r l e c a n i n t h e E C M t o a f f e c t c h o n d r o c y t e f u n c t i o n , a s w a s s h o w n f o r h i s t o n e H 1 i n s k e l e t a l m u s c l e c e l l s ( H e n r i q u e z , C a s a r e t a l . 2 0 0 2 ) . F r o m H D A C 4 k n o c k o u t s t u d i e s , w e g u e s s e d t h a t t h e i n t e r a c t i o n s b e t w e e n p e r l e c a n a n d H 3 a l s o i n v o l v e t h e h i s t o n e d e a c e t y l a s e ( H D A C ) - 4 , w h i c h r e m o v e s a c e t y l g r o u p s , a n d a l s o t h e t r a n s c r i p t i o n f a c t o r R u n x 2 i n c o n t r o l l i n g a s p e c t s o f e n d o c h o n d r a l b o n e g r o w t h ( V e g a , M a t s u d a e t a l . 2 0 0 4 ) .

A T a l e o f T w o G r o w t h F a c t o r s P e r l e c a n h a s d i f f e r e n t r e l a t i v e a f f i n i t i e s f o r F G F - 2 a n d F G F - 1 8 . O u r r e s u l t s i n P a p e r I , F i g u r e 5 w h e n a n a l y s e d w i t h

S i g m a P l o t R e g r e s s i o n W i z a r d g a v e a k d o f 6 5 n M ( F i g u r e 2 3 , b e l o w , p a r t B ) . P a p e r I I r e p o r t s a 1 4 5 n M k d f o r F G F - 1 8 : p e r l e c a n b i n d i n g ( P a r t A ) .

137

F i g u r e 2 3 : D i f f e r r i n g a f f i n i t i e s o f F G F - 1 8 a n d F G F - 2 f o r G r o w t h P l a t e P e r l e c a n .

C o n s i d e r i n g t h e v a r i o u s c a r t i l a g e r e c e p t o r s a n d o t h e r p r o t e i n s t h a t F G F - 1 8 i n t e r a c t s w i t h ( F G F R - 1 , - 2 , - 3 , V E G F a n d V E G F R ) i t i s r e a s o n a b l e t o u n d e r s t a n d w h y F G F - 1 8 i s l e s s t i g h t l y a s s o c i a t e d w i t h p e r l e c a n t h a n F G F - 2 i s . B a s e d o n t h e k n o c k o u t d a t a f o r F G F - 2 ( M o n t e r o , O k a d a e t a l . 2 0 0 0 ) , F G F - 1 8 ( L i u , X u e t a l . 2 0 0 2 ; O h b a y a s h i , S h i b a y a m a e t a l . 2 0 0 2 ; L i u , L a v i n e e t a l . 2 0 0 6 ) , t h e p e r l e c a n G A G s ( R o s s i , M o r i t a e t a l . 2 0 0 3 ) , t o w h i c h F G F - 2 b i n d s , a n d t h e p e r l e c a n c o r e p r o t e i n ( A r i k a w a - H i r a s a w a , W a t a n a b e e t a l . 1 9 9 9 ; C o s t e l l , G u s t a f s s o n e t a l . 1 9 9 9 ) , t o w h i c h F G F - 1 8 b i n d s , w e f u r t h e r p r o p o s e i n t h e d i s c u s s i o n o f o u r d a t a i n P a p e r I I I t h a t F G F - 1 8 i s t h e c r i t i c a l l i g a n d i n e n d o c h o n d r a l b o n e g r o w t h . B i n d i n g t i g h t l y b u t w i t h l e s s a f f i n i t y t h a n F G F - 2 l e a v e s t h i s m o s t c r u c i a l l i g a n d m o r e r e a d i l y a c c e s s i b l e t o r e c e p t o r s a n d o t h e r r e g u l a t o r y p r o t e i n s .

138 A n o t h e r e x p l a n a t i o n f o r t h e d i f f e r e n t a f f i n i t i e s o f t h e t w o g r o w t h f a c t o r s f o r p e r l e c a n m i g h t l i e i n t h e i n c r e a s e d e l e c t r o s t a t i c i n t e r a c t o n b e t w e e n t h e d e n s e l y n e g a t i v e G A G s o f p e r l e c a n a n d t h e h i g h l y b a s i c F G F - 2 v e r s u s t h e i n t e r a c t i o n b e t w e e n t h e F G F - 1 8 a n d d o m a i n I I I o f t h e p e r l e c a n c o r e .

F i n a l l y T a k e n t o g e t h e r , t h e d a t a i n t h i s d i s s e r t a t i o n s h o u l d h e l p t o e x p l a i n p a r t o f t h e d y n a m i c s o f t h e p e r l e c a n i n t e r a c t i o n s s h o w n t o b e s o e s s e n t i a l f o r t h e m u l t i f u n c t i o n a l i t y o f t h i s p r o t e o g l y c a n i n t h e g r o w t h p l a t e . W e h a v e u n c o v e r e d t h e p r e v i o u s l y u n k n o w n r o l e o f t h e C S c h a i n s o n p e r l e c a n a n d h a v e a d d e d k n o w l e d g e o n t h e r o l e s o f t h e H S c h a i n s a n d t h e p e r l e c a n c o r e p r o t e i n i n t h e b i n d i n g o f r e g u l a t o r y p r o t e i n s . T h i s d i s s e r t a t i o n , t h e r e f o r e , i s a n i m p o r t a n t a d d i t i o n t o t h e e x i s t i n g s c i e n t i f i c l i t e r a t u r e o n t h e c o m p l e x r o l e s o f p e r l e c a n i n e n d o c h o n d r a l b o n e g r o w t h .

R e f e r e n c e s f o r C o n c l u s i o n s

A r i k a w a - H i r a s a w a , E . , H . W a t a n a b e , e t a l . ( 1 9 9 9 ) . " P e r l e c a n i s e s s e n t i a l f o r c a r t i l a g e a n d c e p h a l i c d e v e l o p m e n t . " N a t G e n e t 2 3 ( 3 ) : 3 5 4 - 8 .

C h a k r a v a r t i , S . , T . H o r c h a r , e t a l . ( 1 9 9 5 ) . " R e c o m b i n a n t d o m a i n I I I o f p e r l e c a n p r o m o t e s c e l l a t t a c h m e n t t h r o u g h i t s R G D S s e q u e n c e . " J B i o l C h e m 2 7 0 ( 1 ) : 4 0 4 - 9 .

C o s t e l l , M . , E . G u s t a f s s o n , e t a l . ( 1 9 9 9 ) . " P e r l e c a n m a i n t a i n s t h e i n t e g r i t y o f c a r t i l a g e a n d s o m e b a s e m e n t m e m b r a n e s . " J C e l l B i o l 1 4 7 ( 5 ) : 1 1 0 9 - 2 2 .

139 D a v i d s o n , D . , A . B l a n c , e t a l . ( 2 0 0 5 ) . " F i b r o b l a s t g r o w t h f a c t o r ( F G F ) 1 8 s i g n a l s t h r o u g h F G F r e c e p t o r 3 t o p r o m o t e c h o n d r o g e n e s i s . " J B i o l C h e m 2 8 0 ( 2 1 ) : 2 0 5 0 9 - 1 5 .

F a r n u m , C . E . a n d N . J . W i l s m a n ( 1 9 8 9 ) . " C o n d e n s a t i o n o f h y p e r t r o p h i c c h o n d r o c y t e s a t t h e c h o n d r o - o s s e o u s j u n c t i o n o f g r o w t h p l a t e c a r t i l a g e i n Y u c a t a n s w i n e : r e l a t i o n s h i p t o l o n g b o n e g r o w t h . " A m J A n a t 1 8 6 ( 4 ) : 3 4 6 - 5 8 .

G e r b e r , H . P . , T . H . V u , e t a l . ( 1 9 9 9 ) . " V E G F c o u p l e s h y p e r t r o p h i c c a r t i l a g e r e m o d e l i n g , o s s i f i c a t i o n a n d a n g i o g e n e s i s d u r i n g e n d o c h o n d r a l b o n e f o r m a t i o n . " N a t M e d 5 ( 6 ) : 6 2 3 - 8 .

G o v i n d r a j , P . , L . W e s t , e t a l . ( 2 0 0 2 ) . " I s o l a t i o n a n d i d e n t i f i c a t i o n o f t h e m a j o r h e p a r a n s u l f a t e p r o t e o g l y c a n s i n t h e d e v e l o p i n g b o v i n e r i b g r o w t h p l a t e . " J B i o l C h e m 2 7 7 ( 2 2 ) : 1 9 4 6 1 - 9 .

G o v i n d r a j , P . , L . W e s t , e t a l . ( 2 0 0 6 ) . " M o d u l a t i o n o f F G F - 2 b i n d i n g t o c h o n d r o c y t e s f r o m t h e d e v e l o p i n g g r o w t h p l a t e b y p e r l e c a n . " M a t r i x B i o l 2 5 ( 4 ) : 2 3 2 - 9 .

H e n r i q u e z , J . P . , J . C . C a s a r , e t a l . ( 2 0 0 2 ) . " E x t r a c e l l u l a r m a t r i x h i s t o n e H 1 b i n d s t o p e r l e c a n , i s p r e s e n t i n r e g e n e r a t i n g s k e l e t a l m u s c l e a n d s t i m u l a t e s m y o b l a s t p r o l i f e r a t i o n . " J C e l l S c i 1 1 5 ( P t 1 0 ) : 2 0 4 1 - 5 1 .

H u a n g , W . , U . I . C h u n g , e t a l . ( 2 0 0 1 ) . " T h e c h o n d r o g e n i c t r a n s c r i p t i o n f a c t o r S o x 9 i s a t a r g e t o f s i g n a l i n g b y t h e p a r a t h y r o i d h o r m o n e - r e l a t e d p e p t i d e i n t h e g r o w t h p l a t e o f e n d o c h o n d r a l b o n e s . " P r o c N a t l A c a d S c i U S A 9 8 ( 1 ) : 1 6 0 - 5 .

I o z z o , R . V . , I . R . C o h e n , e t a l . ( 1 9 9 4 ) . " T h e b i o l o g y o f p e r l e c a n : t h e m u l t i f a c e t e d h e p a r a n s u l p h a t e p r o t e o g l y c a n o f b a s e m e n t m e m b r a n e s a n d p e r i c e l l u l a r m a t r i c e s . " B i o c h e m J 3 0 2 ( P t 3 ) : 6 2 5 - 3 9 .

K n u d s o n , C . B . a n d W . K n u d s o n ( 2 0 0 1 ) . " C a r t i l a g e p r o t e o g l y c a n s . " S e m i n C e l l D e v B i o l 1 2 ( 2 ) : 6 9 - 7 8 .

L e d b e t t e r , S . R . , L . W . F i s h e r , e t a l . ( 1 9 8 7 ) . " D o m a i n s t r u c t u r e o f t h e b a s e m e n t m e m b r a n e h e p a r a n s u l f a t e p r o t e o g l y c a n . " B i o c h e m i s t r y 2 6 ( 4 ) : 9 8 8 - 9 5 .

140 L i , H . , N . B . S c h w a r t z , e t a l . ( 1 9 9 3 ) . " c D N A c l o n i n g o f c h i c k c a r t i l a g e c h o n d r o i t i n s u l f a t e ( a g g r e c a n ) c o r e p r o t e i n a n d i d e n t i f i c a t i o n o f a s t o p c o d o n i n t h e a g g r e c a n g e n e a s s o c i a t e d w i t h t h e c h o n d r o d y s t r o p h y , n a n o m e l i a . " J B i o l C h e m 2 6 8 ( 3 1 ) : 2 3 5 0 4 - 1 1 .

L i u , Z . , K . J . L a v i n e , e t a l . ( 2 0 0 6 ) . " F G F 1 8 i s r e q u i r e d f o r e a r l y c h o n d r o c y t e p r o l i f e r a t i o n , h y p e r t r o p h y a n d v a s c u l a r i n v a s i o n o f t h e g r o w t h p l a t e . " D e v B i o l .

L i u , Z . , J . X u , e t a l . ( 2 0 0 2 ) . " C o o r d i n a t i o n o f c h o n d r o g e n e s i s a n d o s t e o g e n e s i s b y f i b r o b l a s t g r o w t h f a c t o r 1 8 . " G e n e s D e v 1 6 ( 7 ) : 8 5 9 - 6 9 .

M i n i n a , E . , C . K r e s c h e l , e t a l . ( 2 0 0 2 ) . " I n t e r a c t i o n o f F G F , I h h / P t h l h , a n d B M P s i g n a l i n g i n t e g r a t e s c h o n d r o c y t e p r o l i f e r a t i o n a n d h y p e r t r o p h i c d i f f e r e n t i a t i o n . " D e v C e l l 3 ( 3 ) : 4 3 9 - 4 9 .

M o n g i a t , M . , K . T a y l o r , e t a l . ( 2 0 0 0 ) . " T h e p r o t e i n c o r e o f t h e p r o t e o g l y c a n p e r l e c a n b i n d s s p e c i f i c a l l y t o f i b r o b l a s t g r o w t h f a c t o r - 7 . " J B i o l C h e m 2 7 5 ( 1 0 ) : 7 0 9 5 - 1 0 0 .

M o n t e r o , A . , Y . O k a d a , e t a l . ( 2 0 0 0 ) . " D i s r u p t i o n o f t h e f i b r o b l a s t g r o w t h f a c t o r - 2 g e n e r e s u l t s i n d e c r e a s e d b o n e m a s s a n d b o n e f o r m a t i o n . " J C l i n I n v e s t 1 0 5 ( 8 ) : 1 0 8 5 - 9 3 .

M u e n k e , M . a n d U . S c h e l l ( 1 9 9 5 ) . " F i b r o b l a s t - g r o w t h - f a c t o r r e c e p t o r m u t a t i o n s i n h u m a n s k e l e t a l d i s o r d e r s . " T r e n d s G e n e t 1 1 ( 8 ) : 3 0 8 - 1 3 .

N o o n a n , D . M . , A . F u l l e , e t a l . ( 1 9 9 1 ) . " T h e c o m p l e t e s e q u e n c e o f p e r l e c a n , a b a s e m e n t m e m b r a n e h e p a r a n s u l f a t e p r o t e o g l y c a n , r e v e a l s e x t e n s i v e s i m i l a r i t y w i t h l a m i n i n A c h a i n , l o w d e n s i t y l i p o p r o t e i n - r e c e p t o r , a n d t h e n e u r a l c e l l a d h e s i o n m o l e c u l e . " J B i o l C h e m 2 6 6 ( 3 4 ) : 2 2 9 3 9 - 4 7 .

O h b a y a s h i , N . , M . S h i b a y a m a , e t a l . ( 2 0 0 2 ) . " F G F 1 8 i s r e q u i r e d f o r n o r m a l c e l l p r o l i f e r a t i o n a n d d i f f e r e n t i a t i o n d u r i n g o s t e o g e n e s i s a n d c h o n d r o g e n e s i s . " G e n e s D e v 1 6 ( 7 ) : 8 7 0 - 9 .

O r n i t z , D . M . ( 2 0 0 0 ) . " F G F s , h e p a r a n s u l f a t e a n d F G F R s : c o m p l e x i n t e r a c t i o n s e s s e n t i a l f o r d e v e l o p m e n t . " B i o e s s a y s 2 2 ( 2 ) : 1 0 8 - 1 2 .

141 O r n i t z , D . M . a n d P . J . M a r i e ( 2 0 0 2 ) . " F G F s i g n a l i n g p a t h w a y s i n e n d o c h o n d r a l a n d i n t r a m e m b r a n o u s b o n e d e v e l o p m e n t a n d h u m a n g e n e t i c d i s e a s e . " G e n e s D e v 1 6 ( 1 2 ) : 1 4 4 6 - 6 5 .

O r n i t z , D . M . , A . Y a y o n , e t a l . ( 1 9 9 2 ) . " H e p a r i n i s r e q u i r e d f o r c e l l - f r e e b i n d i n g o f b a s i c f i b r o b l a s t g r o w t h f a c t o r t o a s o l u b l e r e c e p t o r a n d f o r m i t o g e n e s i s i n w h o l e c e l l s . " M o l C e l l B i o l 1 2 ( 1 ) : 2 4 0 - 7 .

P i n e s , M . a n d S . H u r w i t z ( 1 9 9 1 ) . " T h e r o l e o f t h e g r o w t h p l a t e i n l o n g i t u d i n a l b o n e g r o w t h . " P o u l t S c i 7 0 ( 8 ) : 1 8 0 6 - 1 4 .

R i t t e n h o u s e , E . , L . C . D u n n , e t a l . ( 1 9 7 8 ) . " C a r t i l a g e m a t r i x d e f i c i e n c y ( c m d ) : a n e w a u t o s o m a l r e c e s s i v e l e t h a l m u t a t i o n i n t h e m o u s e . " J E m b r y o l E x p M o r p h o l 4 3 : 7 1 - 8 4 .

R o s s i , M . , H . M o r i t a , e t a l . ( 2 0 0 3 ) . " H e p a r a n s u l f a t e c h a i n s o f p e r l e c a n a r e i n d i s p e n s a b l e i n t h e l e n s c a p s u l e b u t n o t i n t h e k i d n e y . " E m b o J 2 2 ( 2 ) : 2 3 6 - 4 5 .

S c h m i e d e k e , T . M . , F . W . S t o c k l , e t a l . ( 1 9 8 9 ) . " H i s t o n e s h a v e h i g h a f f i n i t y f o r t h e g l o m e r u l a r b a s e m e n t m e m b r a n e . R e l e v a n c e f o r i m m u n e c o m p l e x f o r m a t i o n i n l u p u s n e p h r i t i s . " J E x p M e d 1 6 9 ( 6 ) : 1 8 7 9 - 9 4 .

S h i a n g , R . , L . M . T h o m p s o n , e t a l . ( 1 9 9 4 ) . " M u t a t i o n s i n t h e t r a n s m e m b r a n e d o m a i n o f F G F R 3 c a u s e t h e m o s t c o m m o n g e n e t i c f o r m o f d w a r f i s m , a c h o n d r o p l a s i a . " C e l l 7 8 ( 2 ) : 3 3 5 - 4 2 .

S m i t h , S . M . , L . A . W e s t , e t a l . ( 2 0 0 6 ) . " H e p a r a n a n d c h o n d r o i t i n s u l f a t e o n g r o w t h p l a t e p e r l e c a n m e d i a t e b i n d i n g a n d d e l i v e r y o f F G F - 2 t o F G F r e c e p t o r s . " M a t r i x B i o l .

S t - J a c q u e s , B . , M . H a m m e r s c h m i d t , e t a l . ( 1 9 9 9 ) . " I n d i a n h e d g e h o g s i g n a l i n g r e g u l a t e s p r o l i f e r a t i o n a n d d i f f e r e n t i a t i o n o f c h o n d r o c y t e s a n d i s e s s e n t i a l f o r b o n e f o r m a t i o n . " G e n e s D e v 1 3 ( 1 6 ) : 2 0 7 2 - 8 6 .

V e g a , R . B . , K . M a t s u d a , e t a l . ( 2 0 0 4 ) . " H i s t o n e d e a c e t y l a s e 4 c o n t r o l s c h o n d r o c y t e h y p e r t r o p h y d u r i n g s k e l e t o g e n e s i s . " C e l l 1 1 9 ( 4 ) : 5 5 5 - 6 6 .

W a t a n a b e , H . , K . K i m a t a , e t a l . ( 1 9 9 4 ) . " M o u s e c a r t i l a g e m a t r i x d e f i c i e n c y ( c m d ) c a u s e d b y a 7 b p d e l e t i o n i n t h e a g g r e c a n g e n e . " N a t G e n e t 7 ( 2 ) : 1 5 4 - 7 .

142 W a t s o n , K . , R . J . E d w a r d s , e t a l . ( 1 9 9 5 ) . " E x t r a - n u c l e a r l o c a t i o n o f h i s t o n e s i n a c t i v a t e d h u m a n p e r i p h e r a l b l o o d l y m p h o c y t e s a n d c u l t u r e d T - c e l l s . " B i o c h e m P h a r m a c o l 5 0 ( 3 ) : 2 9 9 - 3 0 9 .

W e s t , L . , P . G o v i n d r a j , e t a l . ( 2 0 0 6 ) . " C h a n g e s i n p e r l e c a n d u r i n g c h o n d r o c y t e d i f f e r e n t i a t i o n i n t h e f e t a l b o v i n e r i b g r o w t h p l a t e . " J O r t h o p R e s 2 4 ( 6 ) : 1 3 1 7 - 2 6 .

W h i t e , K . E . , J . M . C a b r a l , e t a l . ( 2 0 0 5 ) . " M u t a t i o n s t h a t c a u s e o s t e o g l o p h o n i c d y s p l a s i a d e f i n e n o v e l r o l e s f o r F G F R 1 i n b o n e e l o n g a t i o n . " A m J H u m G e n e t 7 6 ( 2 ) : 3 6 1 - 7 .

143

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APPENDIX

161

Appendix : Focu s on Per lec an

F o c u s o n M o l e c u l e s : P e r l e c a n ( H S P G 2 )

S i m o n e S m i t h 1 a n d J o h n R H a s s e l l 1 , 2 1 D e p a r t m e n t o f M o l e c u l a r M e d i c i n e , U n i v e r s i t y o f S o u t h F l o r i d a C o l l e g e o f M e d i c i n e , T a m p a , F l 3 3 6 1 2 . 2 C e n t e r f o r R e s e a r c h i n S k e l e t a l D e v e l o p m e n t a n d P e d i a t r i c O r t h o p e d i c s , S h r i n e r s H o s p i t a l s f o r C h i l d r e n – T a m p a , F l . 3 3 6 1 2

K e y W o r d s : b a s e m e n t m e m b r a n e p r o t e o g l y c a n , c a r t i l a g e , h s p g 2 , i n t r a o c u l a r p r e s s u r e

P u b l i s h e d i n E x p e r i m e n t a l E y e R e s e a r c h S e p t e m b e r 2 0 0 6 ; 8 3 ( 3 ) : 4 7 1 - 2 . E p u b M a r c h 2 3 , 2 0 0 6 .

162 A p p e n d i x : ( c o n t i n u e d ) ( 1 ) S t r u c t u r e : P e r l e c a n ( N M _ 0 0 5 5 2 9 ) w a s f i r s t i d e n t i f i e d a s a h e p a r a n s u l f a t e p r o t e o g l y c a n i n b a s e m e n t m e m b r a n e s ( I o z z o 1 9 9 4 ) . I n a r o t a r y s h a d o w e d i m a g e , t h e c o r e p r o t e i n o f p e r l e c a n ( w i t h i t s g l o b u l a r d o m a i n s s p a c e d b y r o d - l i k e s e q u e n c e s ) r e s e m b l e s a s t r i n g o f p e a r l s , h e n c e i t s n a m e ( I o z z o 1 9 9 4 ) . T h e p r o t e i n c o r e i s e n c o d e d b y a n a p p r o x i m a t e l y 1 2 0 k b g e n e w i t h 9 7 e x o n s ( N T - 0 0 4 5 7 6 ) . T h e g e n e i s w e l l c o n s e r v e d a c r o s s s p e c i e s , w i t h h o m o l o g u e s o f m a m m a l i a n p e r l e c a n ( h s p g 2 g e n e ) p r e s e n t i n C . e l e g a n s ( u n c 5 2 g e n e ) , D r o s o p h i l a ( t r o l g e n e ) a n d o t h e r s p e c i e s . I n h u m a n s , p e r l e c a n ’ s c o r e p r o t e i n i s 4 6 6 k D a i n s i z e . M i c e a l s o h a v e a 3 6 9 k D a c o r e p r o t e i n d u e t o a l t e r n a t i v e s p l i c i n g ( H a s s e l l , Y a m a d a e t a l . 2 0 0 2 ) . P e r l e c a n i s a l s o p r e s e n t i n e x t r a c e l l u l a r m a t r i x o f c a r t i l a g e , w h e r e i t h a s b o t h c h o n d r o i t i n s u l f a t e ( C S ) a n d h e p a r a n s u l f a t e ( H S ) c h a i n s ( H a s s e l l , Y a m a d a e t a l . 2 0 0 2 ) . T h e C S a n d H S c h a i n s v a r y i n s i z e a n d s u g a r r e s i d u e s e q u e n c e d e p e n d i n g o n t h e t i s s u e l o c a t i o n o f t h e p e r l e c a n . T h e c o r e p r o t e i n o f p e r l e c a n c o n s i s t s o f f i v e d o m a i n s . D o m a i n I i s t h e m o s t u n i q u e , s h o w i n g s i g n i f i c a n t h o m o l o g y t o n o k n o w n p r o t e i n s ( H a s s e l l , Y a m a d a e t a l . 2 0 0 2 ) . D o m a i n I I s h o w s h o m o l o g y t o p o r t i o n s o f t h e L D L r e c e p t o r , d o m a i n I I I t o r e g i o n s o f t h e l a m i n i n s h o r t a r m , d o m a i n I V t o N - C A M a n d d o m a i n V t o t h e g l o b u l a r d o m a i n o f t h e l a m i n i n A c h a i n . T h e n e g a t i v e l y c h a r g e d g l y c o s a m i n o g l y c a n ( G A G ) c h a i n s a r e a t t a c h e d a t d o m a i n s I a n d V .

163 A p p e n d i x : ( c o n t i n u e d ) ( 2 ) F u n c t i o n : I n c a r t i l a g e , p e r l e c a n i s e s s e n t i a l f o r i n t e g r i t y o f t h e m a t r i x , b i n d i n g p r o t e i n s s u c h a s f i b u l i n a n d f i b r o n e c t i n t o f o r m a s c a f f o l d i n g n e t w o r k b e t w e e n t h e c h o n d r o c y t e s t o o r g a n i z e t h e m a n d s t a b i l i z e t h e c a r t i l a g e ( H a s s e l l , Y a m a d a e t a l . 2 0 0 2 ) . P e r l e c a n a l s o f u n c t i o n s i n c e l l a t t a c h m e n t i n a r e a s s u c h a s b a s e m e n t m e m b r a n e s ( B M ) , w h e r e i t b i n d s t o o t h e r B M c o m p o n e n t s s u c h a s l a m i n i n a n d C o l l a g e n I V ( I o z z o 1 9 9 4 ) . I n s k e l e t a l m u s c l e , p e r l e c a n e n a b l e s m u s c l e c e l l p r o l i f e r a t i o n v i a g r o w t h f a c t o r s i g n a l i n g a n d a f f e c t s c e l l s t a b i l i t y t h r o u g h β 1 - I n t e g r i n s ( I o z z o 1 9 9 4 ) . P e r l e c a n a l s o h e l p s t o m e d i a t e m u s c l e c o n t r a c t i o n b y l o c a l i z i n g a c e t y l c h o l i n e e s t e r a s e t o t h e n e u r o m u s c u l a r j u n c t i o n ( N M J ) s o t h a t m u s c l e s c a n r e l a x a f t e r c o n t r a c t i o n . A c e t y l c h o l i n e a c t s a s a n e u r o t r a n s m i t t e r i n s k e l e t a l m u s c l e , b i n d i n g i t s r e c e p t o r o n t h e c e l l s u r f a c e a n d a c t i v a t i n g s o d i u m c h a n n e l s t o c a u s e m u s c l e c o n t r a c t i o n . A c e t y l c h o l i n e e s t e r a s e r e v e r s e s t h i s p r o c e s s t o c a u s e b o t h m u s c l e r e l a x a t i o n a n d r e c y c l i n g o f t h e n e u r o t r a n s m i t t e r . T h i s e s t e r a s e a n d p e r l e c a n a r e b o t h n o r m a l p a r t s o f t h e c l u s t e r o f p r o t e i n s f o r m i n g t h e N M J . T h e c o l l a g e n - t a i l e d f o r m o f t h e a c e t y l c h o l i n e e s t e r a s e b i n d s p e r l e c a n i n v i t r o a n d i s a b s e n t a t t h e N M J o f p e r l e c a n - n u l l m i c e , s u p p o r t i n g t h e t h e o r y t h a t p e r l e c a n h e l p s t o l o c a l i z e a c e t y l c h o l i n e e s t e r a s e t o t h e N M J . T h e p o l y a n i o n i c G A G c h a i n s o f p e r l e c a n a s w e l l a s i t s c o r e p r o t e i n c a n b i n d g r o w t h f a c t o r s a s d i v e r s e a s F G F 2 , F G F 7 , P D G F a n d E G F t o a f f e c t p r o c e s s e s s u c h a s a n g i o g e n e s i s , c e l l p r o l i f e r a t i o n / d i f f e r e n t i a t i o n , e m b r y o g e n e s i s a n d l o n g b o n e g r o w t h ( I o z z o 1 9 9 4 ) . T h i s m o d u l a t i o n o f c e l l p r o l i f e r a t i o n a n d d i f f e r e n t i a t i o n i s a l s o

164 A p p e n d i x : ( c o n t i n u e d ) i m p o r t a n t f o r t h e r e m o d e l i n g t h a t o c c u r s a f t e r i n j u r y t o t i s s u e s s u c h a s m u s c l e , a r t e r i e s a n d c o r n e a . P e r l e c a n e x p r e s s i o n i s u p r e g u l a t e d a f t e r c o r n e a l s t r o m a l i n j u r y a n d a f t e r a n a r t i f i c i a l i n c r e a s e i n i n t r a o c u l a r p r e s s u r e - w h i c h h e l p s t o m a i n t a i n c o r n e a l s h a p e ( V i t t i t o w a n d B o r r a s 2 0 0 4 ) . P e r l e c a n m u s t , t h e r e f o r e , b e n o t o n l y p r o t e c t i v e i n t h e e y e ( f o r m i n g s t a b l e l e n s b a s e m e n t m e m b r a n e s ) b u t a l s o r e c u p e r a t i v e ( i n r e m o d e l i n g o f i n j u r e d o c u l a r t i s s u e ) . P e r l e c a n m a i n t a i n s c e l l a d h e s i o n a n d i n t e g r i t y o f t h e c o r n e a l m a t r i x ( V i t t i t o w a n d B o r r a s 2 0 0 4 ) , a n d a l s o h e l p s t o m a i n t a i n t h e s e l e c t i v e b a s e m e n t m e m b r a n e b a r r i e r t h a t c o n t r o l s f l u i d f l o w i n t h e e y e ( R o s s i , M o r i t a e t a l . 2 0 0 3 ) .

( 3 ) D i s e a s e I n v o l v e m e n t : P e r l e c a n i s a n e s s e n t i a l m o l e c u l e i n c a r t i l a g e . S i l v e r m a n - H a n d m a k e r t y p e d y s s e g m e n t a l d y s p l a s i a ( D D S H ) i s a “ l e t h a l , a u t o s o m a l r e c e s s i v e s k e l e t a l d y s p l a s i a ” ( H a s s e l l , Y a m a d a e t a l . 2 0 0 2 ) . I t i s a n a l o g o u s t o t h e s e v e r e d w a r f i s m s e e n i n t h e p e r l e c a n n u l l m o u s e . T h e s e m i c e h a v e l i m b a n d s k u l l b o n e s t h a t a r e s h o r t e r a n d w i d e r t h a n n o r m a l . T h e c r a n i o f a c i a l d e f e c t s a r e s e v e r e a n d d e a t h o c c u r s u p t o d a y s a f t e r b i r t h . F o r s o m e o f t h e m i c e , d e a t h o c c u r s p r e n a t a l l y d u e t o d e f e c t i v e b a s e m e n t m e m b r a n e f o r m a t i o n i n t h e a b s e n c e o f p e r l e c a n ( H a s s e l l , Y a m a d a e t a l . 2 0 0 2 ) . T h e r e i s a l s o a d e f e c t i n a c e t y l c h o l i n e e s t e r a s e e x p r e s s i o n a t t h e n e u r o m u s c u l a r j u n c t i o n ( N M J ) o f p e r l e c a n n u l l m i c e . W e r e t h e s e m i c e t o s u r v i v e l o n g e n o u g h , t h e y m i g h t e x h i b i t p a r a l y z e d o r s p a s t i c m u s c l e s t h a t w e r e u n a b l e t o r e l a x a f t e r c o n t r a c t i o n . A d e f e c t i n t h e u n c 5 2 g e n e ( h o m o l o g o u s t o m a m m a l i a n p e r l e c a n g e n e ) i n C . e l e g a n s s i m i l a r l y c a u s e s a d u l t m u s c l e p a r a l y s i s . M u s c l e e x c i t a b i l i t y a n d m y o t o n i a a r e a l s o s e e n i n h u m a n s w i t h D D S H .

165 A p p e n d i x : ( c o n t i n u e d ) P e r l e c a n i s a n i m p o r t a n t c o m p o n e n t o f b a s e m e n t m e m b r a n e s ( B M ) , w h i c h a r e v i t a l i n m a n y t i s s u e s . P e r l e c a n ’ s s t r u c t u r a l ( b a r r i e r ) a n d f u n c t i o n a l ( s i g n a l i n g ) r o l e s i n B M s a r e i n t e g r a l i n t h e e y e s , a s a n o t h e r m o u s e m o d e l s h o w s . M i c e w i t h a t a r g e t e d d i s r u p t i o n i n e x o n 3 o f t h e p e r l e c a n g e n e ( t h e e x o n t h a t e n c o d e s t h e t h r e e G A G a t t a c h m e n t s i t e s o n D o m a i n I o f p e r l e c a n ) h a s a b n o r m a l e y e s ( R o s s i , M o r i t a e t a l . 2 0 0 3 ) . T h e e y e s o f t h e s e h s p g 2 ∆ 3 / ∆ 3 m i c e a r e 7 7 % t h e w e i g h t o f n o r m a l e y e s , d u e t o a p o p t o s i s . T h e l e n s e p i t h e l i a l c e l l s o f t h e p e r l e c a n m u t a n t s s h o w n o r m a l p r o l i f e r a t i o n . H o w e v e r , w h e n t h e s e m u t a n t s a r e c r o s s - b r e d w i t h C o l X V I I I m u t a n t s , t h e p r o l i f e r a t i o n o f t h e l e n s e p i t h e l i a l c e l l s i s a b n o r m a l . T h i s d i f f e r e n c e i n d e f e c t i s l i k e l y d u e t o h a v i n g c o m p e n s a t o r y C o l X V I I I h e p a r a n s u l f a t e c h a i n s s t i l l p r e s e n t a n d f u n c t i o n a l ( b i n d i n g g r o w t h f a c t o r s ) i n t h e p e r l e c a n m u t a n t s b u t l o s t i n t h e d o u b l e m u t a n t s . T h e l e n s f i b e r s i n t h e h s p g 2 ∆ 3 / ∆ 3 m u t a n t s f o r m n o r m a l l y a t f i r s t b u t g r a d u a l l y s w e l l , b e c o m i n g a m o r p h o u s a n d r u p t u r i n g t h e l e n s b y t h e t i m e t h e m i c e r e a c h 6 m o n t h s o f a g e . T h i s s w e l l i n g i s d u e t o a n o t h e r r o l e o f p e r l e c a n i n t h e l e n s c a p s u l e w h e r e p e r l e c a n m a i n t a i n s a s e l e c t i v e b a r r i e r b e t w e e n t h e v i t r e o u s b o d y a n d t h e l e n s , b i n d i n g t o m o l e c u l e s v i a i t s G A G c h a i n s a n d a i d i n g t h e s e l e c t i v e e x c h a n g e o f g r o w t h f a c t o r s , m e t a b o l i t e s a n d n u t r i e n t s . W i t h t h e p e r l e c a n G A G s a b s e n t , t h e f l o w f r o m t h e l e n s a n d o t h e r r e g i o n s g o e s u n c h e c k e d , w i t h t h e f i b e r s s w e l l i n g d u e t o a b n o r m a l l y h i g h f l u i d i n t a k e . T h e r e a r e a l s o e y e a n o m a l i e s i n t h e r e p e r t o i r e o f h u m a n d i s o r d e r s c a u s e d b y d e f e c t i v e p e r l e c a n . A D D S H f e t u s h a s b e e n r e p o r t e d w i t h b i l a t e r a l c a t a r a c t s a n d e x o p h t h a l m o s ( a b n o r m a l

166 A p p e n d i x : ( c o n t i n u e d ) p r o t r u s i o n o f t h e e y e b a l l ) . I n t h e m i l d e r p e r l e c a n - r e l a t e d S c h w a r t z - J a m p e l S y n d r o m e ( S J S ) , p a t i e n t s s u f f e r m y o p i a o r ‘ n e a r - s i g h t e d n e s s ’ ( H a s s e l l , Y a m a d a e t a l . 2 0 0 2 ) . W i t h t h e w i d e e x p r e s s i o n o f p e r l e c a n i n t h e e y e , i t i s e a s y t o s e e h o w t h i s p r o t e o g l y c a n c a n b e i n v o l v e d i n d i s e a s e s t h a t c a u s e d e f e c t i v e v i s i o n .

( 4 ) F o c u s o f F u t u r e S t u d i e s : H u m a n d i s o r d e r s a n d a n i m a l m o d e l s a r e i n t r i g u i n g t a r g e t s f o r f u r t h e r s t u d i e s o f t h e f u n c t i o n s o f p e r l e c a n i n t h e e y e . S J S a n d D D S H p a t i e n t s p r e s e n t a w i d e s p e c t r u m o f p h e n o t y p e s . I n D D S H a n d t h e l e s s s e v e r e S J S , e y e s c o u l d b e r o u t i n e l y e x a m i n e d f o r d e f e c t s . T e s t s c o u l d m i r r o r t h o s e d o n e w i t h t h e h s p g 2 Δ 3 / Δ 3 m o u s e , t h a t i s , a s s e s s i n g l e n s i n t e g r i t y , o c u l a r b a s e m e n t m e m b r a n e f o r m a t i o n a n d a n y e v e n t u a l e y e d e g e n e r a t i o n c a u s e d b y t h e l a c k o r a b s e n c e o f f u n c t i o n a l p e r l e c a n . A n o t h e r a p p r o a c h f o r f u t u r e s t u d i e s i s t o i n v e s t i g a t e d i s e a s e s t h a t i n v o l v e a v i s u a l d e f e c t b u t h a v e n o t b e e n l i n k e d t o p e r l e c a n . B i o c h e m i c a l a n d m o l e c u l a r a n a l y s i s o f p e r l e c a n i n t h e s e p a t i e n t s c o u l d h i g h l i g h t a n y r o l e f o r p e r l e c a n i n t h e s e d i s o r d e r s . F o r e x a m p l e , o n e n c o u n t e r i n g i n d i v i d u a l s w i t h m u s c u l a r d y s t r o p h y o r s o m e o t h e r c o n g e n i t a l d i s e a s e a n d w h o h a v e d e f e c t i v e v i s i o n , a n a l y s i s o f p e r l e c a n e x p r e s s i o n i n t h e i r o c u l a r t i s s u e s w i l l d e t e r m i n e t h e p a t t e r n o f p e r l e c a n e x p r e s s i o n i n t h e s e d i s o r d e r s . I n s u c h s t u d i e s , c o r r e l a t i o n s b e t w e e n t h e p e r l e c a n g e n o t y p e o f p a t i e n t s a n d t h e e y e p h e n o t y p e s t h e y h a v e c o u l d f u r t h e r u n c o v e r t h e o c u l a r f u n c t i o n a l i t y o f t h i s m u l t i f a c e t e d p r o t e o g l y c a n .

167 A p p e n d i x : ( c o n t i n u e d ) F i g u r e 2 3 .

P e r l e c a n c o r e p r o t e i n : D e t e r m i n e d d o m a i n s t r u c t u r e a n d G A G a t t a c h m e n t . D o m a i n s I I t o V o f p e r l e c a n s h o w h o m o l o g y t o t h e L D L r e c e p t o r ( L D L r ) , t h e s h o r t a r m o f l a m i n i n A a n d B , t h e n e u r a l c e l l a d h e s i o n m o l e c u l e ( N C A M ) , a n d t h e g l o b u l a r a r m o f l a m i n i n A a n d B . T h e h e p a r a n s u l f a t e o r c h o n d r o i t i n s u l f a t e G A G s a r e a t t a c h e d o n d o m a i n s I a n d V . A l s o d e p i c t e d i s t h e r e g i o n o f d o m a i n I t h a t i s a b s e n t w h e n e x o n 3 i s d i s r u p t e d .

R e f e r e n c e s F o r A p p e n d i x

H a s s e l l , J . , Y . Y a m a d a , e t a l . ( 2 0 0 2 ) . " R o l e o f p e r l e c a n i n s k e l e t a l d e v e l o p m e n t a n d d i s e a s e s . " G l y c o c o n j J 1 9 ( 4 - 5 ) : 2 6 3 - 7 .

I o z z o , R . V . ( 1 9 9 4 ) . " P e r l e c a n : a g e m o f a p r o t e o g l y c a n . " M a t r i x B i o l 1 4 ( 3 ) : 2 0 3 - 8 .

V i t t i t o w , J . a n d T . B o r r a s ( 2 0 0 4 ) . " G e n e s e x p r e s s e d i n t h e h u m a n t r a b e c u l a r m e s h w o r k d u r i n g p r e s s u r e - i n d u c e d h o m e o s t a t i c r e s p o n s e . " J C e l l P h y s i o l 2 0 1 ( 1 ) : 1 2 6 - 3 7 .

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About the Autho r

Simone Mar sha-L ee Smith wa s born an d rais ed in J ama ica t hen immigrat ed wit h her famil y to Florid a at the a ge of s ixte en. She gradu ated from th e Uni ver sity of Sout h Florida wi th Univ er sity Honor s, a Ba ch elor of Sci enc e in Bi olo gy and a mi nor in Psyc holo gy. She co mple ted a n und ergr adua te re se arch th es is w ith Dr. William G ower at USF. Her un derg raduat e ex perie nc e fost ered in her a lov e for res ear ch, whic h cont i nued on h er enrol lmen t in USF’s IP 2 C M B (Interdi sci plin ary Ph. D Program in C ellul ar and Mol ecul ar Bio logy) in th e Ins titut e for Biomol ecu lar Sci enc e (IBS). Her ten ure with Dr. Has s ell an d his v a rious l ab memb er s over th e year s ha s promo ted h er conti nue d com mitment to r es earc h.