Expression of osteopontin in developing mouse brain

전체 글

(1)³¤¥Èó. * .* a. (2004) 44 3 Korean J Vet Res(2004) 44(3) : 335~341. *

(2) %Lòö*~PTUFPQPOUJO**. BºÁFÁ^?«ÁÁ¶z Áæz*

(3). *L ³ë« >~ ÏÎL >~ (²Òß: 2004j 8ú 9¢) 1. Expression of osteopontin in developing mouse brain Gyubeom Kim, Insun Hwang, Changjong Moon, Taekyun Shin, Hwa-young Son1 and Youngheun Jee* Department of Veterinary Medicine, College of Agriculture and Life Science, Cheju National University, Jeju 690-756, Korea 1 College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, Korea (Accepted= August 9, 2004) Abstract : This study was undertaken to examine the developmental expression of osteopontin(OPN) in the mouse brain. In Western blotting analysis, the expression of OPN was noted initially at embryonic stage and increased gradually after birth and decreased at postnatal day 60(P60). In immunohistochemistry, OPN expression was found in the interstitial nucleus Cajal and the substantia nigra reticularis in anterior part of the brain and in the inferior olivary complex, the parabrachial nucleus, the facial nucleus, the gigantocellular reticular nucleus, the trigeminal nucleus and the anterior interposed nucleus in posterior part of the brain at P31 and P60. In addition, OPN expression in widespread neurons appeared during the period of neuronal differentiation, increased just after birth and decreased with maturation. These results suggest that OPN contributes to developmental processes, including the differentiation and maturation of specific neuronal populations. Key words : Osteopontin, developing brain, mouse. * . f RGD¢ B αvβ1, αvβ3, αvβ5 rÖ" ç^ ·Ï~ «~~ ^ö ¦O > ® > , RGD¢ B ~æ pz¢ê CD44f α8β1, α9β1 rÖ" ç^·Ï~ «~~ ^ö ¦O > ®º ©b rJ^ ® [3]. "öº f ?f OPN~ 2^öB~ VË öê "Ã>w öB VËj ¾æÞ º . ® . ^, 2^, Wz T ^, RF Î^, çb^, .&ï"^, ¶Ú^ ô f «~~ ^ f ÒÆ, Ã¶ $º "Ã. Bbîö ~ Wö >w~ OPNj B* r J^ ® [6]. $ #çãz6 [18], ^&*îWR Fz [7], Ö" öÃ~ Gj«;W [14], ³F{Ë F~ neointimal formation [5], metastasizing tumor [9], òãï [20] " ?f · ÷Òb' çöB. f ªjW Wb *öº b ®ÒVê ®. f ¾rö .öB ªÒ>î b ÚB Ë" V&î£ï Ú «·ç Ò > .& j ~º · çböBê B Ò>î. ¾r ªÒB .öB~ VË f 2^ö ®º rÖ F~º ¦O W jÖ B ¢ b Úæº ©b rJ^ ® .öB 2^ º ¶~ ^ïö ®º 7Oª¶ rÖj Ï ÞÓÊö Ò~º OPN ~ RGD Vj b > VËj >¯ [13]. öê OPN Osteopontin(OPN) bone sialoprotein I, urinary stone protein, spp-1, early T lymphocyte activation 1 protein, nephropontin, uropontin [1]. OPN , , , , , [4]. OPN OPN , OPN Arg-Gly-Asp(RGD) [12].. \º 2002j *"L OÒFwÏ\² . \j~ æöbR ÚHr.. *Corresponding author: Youngheun Jee Department of Veterinary Medicine, College of Agriculture and Life Science, Cheju National University, Jeju 690-756, Korea [Tel: +82-64-754-3374, Fax: +82-64-756-3354, E-mail: [email protected]]. 335.

(4) BºÁFÁ^?«ÁÁ¶zÁæz. 336. º OPN B* Ã&B B : ® . VF :f ? OPN B"; 7ö öB ~ ^7O Wb~ VË" z®Ú C²z ö V~, êöB~ ^ 7O, " ö 7º j ~º ©b B'b B :& ®b¾, ãêöB OPN ÚÊ j ¾æÚºæö &Bº B :& ~ ì . V¢B öBº B 7 ö ®º v~ ãêöB OPN~ B* ·çj r¶ ~& . f ?f ;¢ Û B~ ®¾ W?B òöB OPN ÚÊ VË' ~¢ <ºæö & r > ®j © . öBº OPN jV 5 Â ê v~ ò öB *~ ã"ö V B* ·~ Nf B* ¦* ¢ rjV * Western blotting" "ï, Ò ;7"ïj Ï~& .. Òò 5 O». þÿ> `ç¾Ò öB ÒÏ ICR vº & þÿböB . ~&b, þÿb ÒG V&ö ~~ 22Û1 C~. ÚNêf 12* "V~ «j Fæ~& þÿb Ï ÆAÒò(Â~B¢Òò)f bf ¶F S~ê ~ ÒG~& . jV þj * ;{ ªV *j r ®º z" · _(jV 17¢, Â ê 3, 7, 14, 21, 31, 60¢_)~ v C 24îÒ& ÒÏ >î . þö B ÿbf ör î~ ª 7 Ú¢ ~ j¢ áî, Â ê ' ¢_ ê ¦¦~& . BV 5 WËV~ òçöB~ OPN B* ;ê¢ rjV * òçj j~ Wî ªCj * -70 C ïÿ &~& . Â ê 31¢ 5 60¢~ òº ò *>¦(ê'Þ 5 &ò>, ~ ò *>¦ Û)f ¾^æö ~º ò ê>¦(7 ò, ²ò, òv 5 >, ~ ò ê>¦ Û) ¾* Ú &~& . Ò BF¾& ËVöB OPN ~ B* ·çj jv~V * ÚV#W F¾ *Ëj j~ òf ?f O»b &~ þö ÒÏ~ & . $ òöB~ OPN B* ·çj &V~V * òçj j~ 20% 7WöÖö ;~, ò *>¦~ ã(ê'Þ 5 &ò>~ *>¦, ~ ò *>¦~ ãb Û)" ò *>¦~ Êã(&ò> ~ ê>¦, ~ ò *>¦~ Êãb Û), Ò ò ê>¦ ¾*Ú Ò; ê, Ûç' O»ö V¢ 2 ¢f ~ ³.Þj ò î . -² ò Úê ³.Þf ê'Þ¦V BBö V¢ *vb . Þj ò Ú ®^¢ Z rö, ¢; *Ïê ¢ o. o. ÚÚ þö ÒÏ~& . `çz ï # ;% ï òöB OPN~ ª 5 B* ·çj &V~V * çz "ïj ~& . çz "ïf avidin-biotin complex Elite kit(Vector, Birlingham, CA)¢ Ï~& . ç.Þf 2¢fj B ê 0.3% "Ö z>²öB '~ ÚW peroxidase¢ B~& . ê jß' >wj Oæ~V * blocking horse serumj >wV . çÚ OPN B*j {~V * OPN(1:300; Santa Cruz Biotechnology, Inc)j Nö B 1* >wÎ ê biotinylated anti-mouse IgG(Vector, Birlingham, CA)¢ NöB 45ª ÿn >wV . ÚB avidin-biotin peroxidase conjugate(Vector, Birlingham, CA) 45ª* NöB >wVb >w Â çf 3, 3'-diaminobenzidine(DAB; Vector, Birlingham, CA) ÏöB BïV . ' ê Òöº PBS Ï ª® ^¿~& . ·W>wj ¾æÞ çj hematoxylin Ïb &"ï~, ethanol, xylene~ î> 5 R« z ";j ê /«~ 7*ã~öB &V~ & . $ çz "ïöB~ Î²>wö ~ jß ' "ïj 6ê~V * ;7 "ïj ~&. . ;7 "ïf çz "ï O»" ÿ¢~ ¾, 2N Úº Cy3-conjugated anti-mouse IgG antibody (1:50; Serotec, Ltd)¢ ÒÏ~, NöB j N ç 45ª* >wV . ê PBS 5ª* 3² ^ ¿ ê z öB ;7 *ãb &V~& . ;7 æº Fluoview FV500 software(Olympus, Co)¢ Ï ~ áî . 8FTUFSO CMPU BOBMZTJT Wîf 1 mM phenylmethylsulfonyl fluoride, 10 mg/ µl leupeptin, 2 mM sodium orthovanadate, 10 µg/ml aprotininj F 20% SDS Ïj Ï ºÂ~ & . ºÂB Wîj ;ï~, sodium dodecyl sulfatepolyacrylamide gelö Æ ê ÿ¢ ·~ Wîj * V'ÿ ~& (7.5% SDS-PAGE). ªÒB Wîf nitrocellulose membranesö 100 V 2* ÿn transfer ~& . êö 5% skim milk¢ ÒÏ~ 1* ÿn. NöB blockingj ~& . OPN(1:1000)j 1N Ú ÒÏ~ NöB 1* >wÊ, 2N Úº HRP-conjugated anti-mouse IgG(1:250; Santa Cruz Biotechnology, Inc)¢ ÒÏ~ NöB 45ª* >w V . Protein bandsº z öB chemiluminescence reagent (LumiGLO, Cell Signaling Technology, Inc) B*j .

(5) B

(6) 7 v òöB~ osteopontin B*. V '. ~ &êº. scanning laser densitometer(GS. band 700, Bio-Rad, Hercules, CA) , Molecular Analyst software(Bio-Rad, Hercules, CA) .. C~&. ¢ Û áîb ¢ Ï~ ª. Ö 8FTUFSO CMPU BOBMZTJT B 7 v òöB~ WËö V OPN~ B* ·j rjV * Western blot analysis¢ ÛB v~ ò~ OPN W·~ æz¢ &V~& . òöBº jV 17¢, Â ê 3, 7, 14, 21, 31, 60¢öB {~ & . OPN~ ª¶ïf post-translational modificationö ~ 44-75 kDab ¾æÂ >Ú ®b [2, 15], þöBº £ 75 kDaöB major band& ¾æÒb £ 55 kDaöB minor band& &V>î . £ 75 kDa band º jV(17¢)öB ¾æÒ(Fig. 1A, B, lanes 1-2), Â çê Ã&~ 3¢(Fig. 1A, B, lane 3), 7¢(Fig. 1A, B, lane 4), 14¢(Fig. 1A, B, lane 5), 21¢(Fig. 1A, B, lane. Fig. 1. Developmental profiles of OPN in the mouse brain (A), and liver (C). The bands show approximately 75 kDa in this study. Total protein was extracted from each organ at the indicated ages. B, D: The intensity of the OPN protein bands shown in A and C was determined by densitometric analysis. E, embryonic days; P, postnatal days; a, anterior part of the brain; p, posterior part of the brain.. 337. ¢ ræ Fæ~& ¾ ¢öBº * BV 6²~º ãËî. Â ê ¢öBº ò ê>¦~ B* · ò *>¦~ B* ·ö j £* Ã& >Ú ®î. 6² ãËj ¢öBº ò *>¦f ò ê>¦~ B* ·ö ~ N & ìî. ²ê £ º jV ¢ öBò ;~² ¾æÒ & êº £~² Ò~&. $ òöB~ B* ·" jv~V *. þ BF¾& ÚV#W F¾~ *ËöB j V ¢" Â ê 5 ¢~ B* · j { Ö" £ º jV ¢ ¦V £~² Ò~& Ò Â ê ¢ ¢ ¢ öBº jVö j ç&'b Ã &~& & Â ê ¢ öBº 6²~& Þ j Ï öB ' Vê ·' Nº &V > ìî. `çz ï # ;% ï Western blot analysisöB B*· &Ë Ã&>î~ 31¢_" B*· 6²>î~ 60¢_öB Â ê 31 ¢ 5 60¢_ v~ òöB OPN B* ·çj {~ V * çz "ïj ~& . Â ê 31 ¢ 5 60¢_~ v~ ò *>¦~ ¦ªöBº ·W >wj &V > ìì, ò *>¦~ êOö * ~ö ®º ¾*î(interstitial nucleus Cajal)" wïî b¦ª(substantia nigra, part reticularis)öB OPN £~² B*~& . Â ê 60¢öBº, 31¢ v~ ò *>¦~ ÊãöB ¾æÒ~ ¾*î" wïî b¦ª 7öB ¾*îöBò OPN £~² B*~&, wïî b¦ª~ OPN B*f &V> æ p~ . Ò Â ê 31¢ ò ê>¦öBº j O(parabrachial nucleus)(Figs. 2B; 3A~ zÚ^Ò; 3D~ zÚ^Ò)" ~RÒ2 Ú(inferior olivary complex)(Figs. 2D; 3C~ zÚ^Ò, 3F~ zÚ^Ò; 3B~ zÚ)öB ; ·W >w &V>î . Ò nã(facial nucleus)(Figs. 2C; 3B~ zÚ^ Ò; 3E~ zÚ^Ò), &^b(gigantocellular reticular nucleus), âNã(trigeminal nucleus)öBº 7ê~ ·W >w &V>î . Þ, 60¢_öBº 31¢_öB OPN B*>î~ jO, nã, &^b, âNãöBº OPN B*~æ p~. . Ò 60¢öBº 31¢ ·W>w &V>î ~ ~RÒ2 ÚöB *® ·W>wj &,. 6), 31 (Fig. 1A, B, lanes 7-8) . 60 (Fig. 1A, B, lanes 9-10). 31 OPN (Fig. 1A, B, lanes 7-8). 60 (Fig. 1A, B, lanes 9-10). 55 kDa band (17 ) . OPN , 17 14, 21, 31 60 OPN , 75 kDa band 17 (Fig. 1C, D, lanes 1-2) . 14 (Fig. 1C, D, lane 3), 21 (Fig. 1C, D, lane 4), 31 (Fig. 1C, D, lane 5) 60 (Fig. 1C, D, lanes 6-7) . β-actin Western blotting ..

(7) 338. BºÁFÁ^?«ÁÁ¶zÁæz. Fig. 2. Schematic diagram of parabrachial nucleus (PB of B), facial nucleus (7N of C) and inferior olivary complex (IO of D) showing OPN-positive reaction in the mouse brain. A: Sagittal section of the mouse brain. Line b, c, d indicate anteroposterior locations of B(5.40 mm), C(5.88 mm), D(6.96 mm). (George Paxinos and Keith Franklin. The Mouse Brain in Stereotaxic Coordinates. Academic Press. 2000).. Fig. 3. Immunostaining for OPN in the P31 midbrain. A: A arrowhead points the parabrachial nucleus(×13.2). B: A arrowhead points the facial nucleus(×13.2). C: Arrowheads point the inferior olivary complex(×13.2). D, G: Higher magnification of A (×66, ×132). E, H: Higher magnification of B(×66, ×132). F, I: Higher magnification of C(×66, ×132)..

(8) B

(9) 7 v òöB~ osteopontin B*. 339. Fig. 4. Immunofluorescencestaining for OPN in the P31 midbrain. A: A arrowhead indicates the parabrachial nucleus (×66). B: Arrowheads indicate the facial nucleus(×66). C: Arrowheads indicate the inferior olivary complex(×66). D: Higher magnification of C(×132).. ¢öBº &V>æ p~~ ã7**~(anterior öB £ ·W>w &V>î . ~ B* ;¢ 2k~V * VNöB ·W> wj ãj &V~, OPN ãö ^Ú~ ^îöB "ã; ¾æ¾ ®rj { > ®. (Fig. 3G, H, I). $ çz "ïöB~ Î²>wö ~ jß ' "ïj 6ê~V * ;7 "ïj ~&. . ;7 "ïj Ï Â ê 30¢ v~ ò öB OPN B* ·çj {~&º, OPNf ç z "ïöB~ Ö"f ¢~~² ò ê>¦~ jO (Fig. 4A~ zÚ^Ò), nã(Fig. 4B~ zÚ^ Ò), ~RÒ2 Ú(Fig. 4C~ zÚ^Ò; 4D~ z Ú^Ò)öB B*~& . V¢B, ò ê>¦~ >~ ã öB OPN B* ·çj &V~&, º Western blottingöB~ Ö"f FÒ ãËWj & .. 31 interposed nucleus) OPN. V. *Òræ ãêöB OPN &æº ª' .VË ' ~ö & B :º ôæ pb, &ç ê ²>~ ÿb «ö ;>Ú ®î . öBº B~º v òöB OPN~ B* ·çj rV * BVö V OPN B* ·~ æzf OPN B* >º ¦*¢ &V~& . çz "ï~ Ö"ö B r > ®, vöBº ò *>¦ º ò ê> ¦öB OPNj B*~º ã z ô ¾æÒ. . $ B 7 v ò~ Western blotting Ö"ö Bê ò *>¦ ò ê>¦~ OPN B* · z ô &V>î . vöB~ ·çf ÑöBê FÒ~² ¾æ¾º ©b >î [10]. B 7 ÑöBº ãêÛ ªz~º ÿn .V ¿Ó" ê òöB OPN~ B* &V>î º & ® [19]. Ò, ê~ ¢; BG V* ÿnöº 7ò, òv, .

(10) 340. BºÁFÁ^?«ÁÁ¶zÁæz. >, ²òö ®º >~ ãöB OPN mRNA& B *~V ·~&b, OPN mRNA~ B*f * æ Îö V¢ 66 Ã&~& [10]. $ in situ hybridizationj Ï W? ÑöB~ º ²ò f ò*~ ß; ã÷öBò OPN mRNA~ 7º * B* &V>, «òf *òöBº OPN mRNA ~ B* ¾æ¾æ p~rj & [17]. V¢B, Ö" f vöB OPN~ B* ¦*& ò *>¦ º ò ê>¦ö ÷7>Ú ®b, OPN ò ê> ¦~ B";ö 7º j ©¢º ©j B . 31¢_~ v ò ê>¦ ~RÒ2 Ú~ ã ööB OPN B* &V>î, Ìf V 60 ¢_öBº 31¢_öBfº Ò ò ê>¦~ ã7* *~öB OPN B* &V>î . f ? OPNf v òöB BVö V¢ B ãöB B *~& . $ vöB OPNf nã 5 âN ã " ? VË'b · ¦*öB B*~& º, ÑöBê f FÒ ·ç &V>î º & ® [10]. WË ÑöB ¯ê *~ . f 7ò U ® ~ ¢N6'ãö" *;, Ó', âN, V"ã." ? VË'b · ¦*öB OPN B*Nj & [8]. º Ö OPN v ~ òöB · VËj &æ, v 7ºãê B ~ .ö V ©ªj B & . Western blot analysis Ö"öBº v& WË~º 7 öº Ã&~~ OPN B* WË jò>º VöB º 6²~& . ¾ ÑöB OPN~ B*·çö & B :ö ~~ ÑöBº WË> OPN mRNA~ B* Ã&~, ·ç FæB . [10]. f ? W? vf Ñ*~ OPN B* · çf ç N¢ & . ¢ öB ;{® « >º ìb¾, &ç ª¶ >&(mRNA $º Wî)~ N, $º vf Ñ~ « * Nö V ©¢ >ê ®j © . OPNf RGD¢ B αvβ1, αvβ3, αvβ5 rÖ" ç^·Ï~ «~~ ^ö ¦O > ® >, RGD¢ B ~æ pz¢ê CD44f α8β1, α9β1 rÖ" ç^·Ï~ «~~ ^ö ¦ O > ®º ©b rJ^ ® [3]. WË~º, Ò W? Ñ ò~ OÒç v^f ò~ · ¦ *öB αv-rÖ mRNA& B*6 º ê ®î. [16]. ~æò ;ç òçöB ¾æ¾º αvβ3 $º CD44~ B*f jç ;{® >æ á~ ®b, ãööB &V>º OPN" rÖ >ÏÚ ~ Ö , $º CD44f~ Ö B~º VË «{®. >æ á~ ® [10]. òöB OPN" Ö~º. · rÖ" CD44, Ò ~ Ö ¾æÚ º VËö &Bº b z >Ú¢ © . VË'b OPNf ^, ï"^, Úb^ , jv^ö z"Wj &ê >î [6, 11]. ßê®, ²' ò î. êö OPN ãjv^f ^¢ ~º ^ ~ "f Wzö V ~, ^*î~ Ò;Wöê & ©¢º & ®î [20]. ßê î~ ìº ¢>' çöBº ãööBò &V>~ OPN, ò î." ?f ÷Ò ' çöBº ãjv^f ^ Ò ^ *îöBê &V6 º Ò f Ö . òöB ~ OPN~ ;{ VËj «~V *Bº ãö ö ò jî¢, ªjB OPN Wî Ö~º ^. ö & ê jº © . º Â *" Â ê, WË~º v ò~ OPN B* ·" ª¢ ^' >&öB ¾rb & . öB Ú Ò f, OPN B* ã^& ªz~º ÿn ·>î º ©", B* v~ WË" þ Ã&~ & WË jò>º 6öBº 6²® º © . æ OPNf WË~ º v~ òöB, ß; ãö ÷~ ªzf W?j ~º B";ö 7º VËj ~Ò¢º ©j B & .. Ö . º B 7 v~ òöB osteopontin(OPN) B* ·ç~ æz¢ rV * >¯>î . Western blot analysisö ~B òöB~ OPN B*f jVö &V >î, Â ê 6N Ã&~ & 60¢_öBº 6²~ º ãËî . Ò òöB~ B* ¦*¢ rjV * çz "ïöB, Â ê 31¢ 5 60¢_~ ò *>¦öBº ¾*î(interstitial nucleus Cajal)" wïî b¦ª(substantia nigra, part reticularis)~ ãööB OPN B* ¾æÒ, ò ê >¦öBº ~RÒ2 Ú(inferior olivary complex), jO(parabrachial nucleus), nã(facial nucleus), &^b(gigantocellular reticular nucleus), âN ã(trigeminal nucleus) 5 ã7**~(anterior interposed nucleus)~ ãööB OPN B* &V>î. . f ? ò~ *>¦öB ò ê>¦~ >~ ã öB OPN B* ·çj ¾æÚº ãËî . ãööB~ OPN B*f ã^~ ªz& ¢ Ú¾º Vö ¾æ¾, Â çê Ã&~ &, WË jò>º VöBº 6²~º ãËj ¾æÞ . .

(11) B

(12) 7 v òöB~ osteopontin B*. æ Ö" f v~ òöB OPN ß; ãö ÷~ ªzf W?j ~º B";ö V ©b ÒòB .. ^^ò 1. Butler, W. T. The nature and significance of osteopontin. Connect. Tissue. Res. 1989, 23, 123-136. 2. Butler, W. T. Structural and functional domains of osteopontin. Ann. N. Y. Acad. Sci. 1995, 760, 6-11. 3. Chellaiah, M. A. and Hruska, K. A. The integrin alpha(v)beta(3) and CD44 regulate the actions of osteopontin on osteoclast motility. Calcif. Tissue. Int. 2003, 72, 197-205. 4. Davis, R. L., Lopez, C. A. and Mou, K. Expression of osteopontin in the inner ear. Ann. N. Y. Acad. Sci. 1995, 760, 279-295. 5. Giachelli, C. M., Bae, N., Almeida, M., Denhardt, D. T., Alpers, C. E. and Schwartz, S. M. Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques. J. Clin. Invest. 1993, 92, 1686-1696. 6. Giachelli, C. M., Liaw, L., Murry, C. E., Schwartz, S. M. and Almeida, M. Osteopontin expression in cardiovascular diseases. Ann. N. Y. Acad. Sci. 1995, 760, 109-126. 7. Giachelli, C. M., Pichler, R., Lombardi, D., Denhardt, D. T., Alpers, C. E., Schwartz, S. M. and Johnson, R. J. Osteopontin expression in angiotensin II-induced tubulointerstitial nephritis. Kidney. Int. 1994, 45, 515-524. 8. Ichikawa, H., Itota, T., Nishitani, Y., Torii, Y., Inoue, K. and Sugimoto, T. Osteopontin-immunoreactive primary sensory neurons in the rat spinal and trigeminal nervous systems. Brain. Res. 2000, 863, 276-281. 9. Khokha, R., Waterhouse, P., Lala, P., Zimmer, M., Denhardt, D. T. and Khokka, R. Increased proteinase expression during tumor progression of cell lines downmodulated for TIMP levels: a new transformation paradigm? J. Cancer. Res. Clin. Oncol. 1991, 117, 620. 10. Lee, M. Y., Choi, J. S., Lim, S. W., Cha, J. H., Chun, M. H. and Chung, J. W. Expression of osteopontin mRNA in developing rat brainstem and cerebellum. Cell. Tissue. Res. 2001, 306, 179-185. 11. Liaw, L., Almeida, M., Hart, C. E., Schwartz, S. M. and Giachelli, C. M. Osteopontin promotes vascular. 12.. 13.. 14.. 15.. 16.. 17.. 18.. 19.. 20.. 341. cell adhesion and spreading and is chemotactic for smooth muscle cells in vitro. Circ. Res. 1994, 74, 214224. Liaw, L., Lindner, V., Schwartz, S. M., Chambers, A. F. and Giachelli, C. M. Osteopontin and beta 3 integrin are coordinately expressed in regenerating endothelium in vivo and stimulate Arg-Gly-Aspdependent endothelial migration in vitro. Circ. Res. 1995, 77, 665-672. Nakamura, I., Rodan, G. A. and Duong le, T. Regulatory mechanism of osteoclast activation. J. Electron. Microsc. (Tokyo). 2003, 52, 527-533. Nau, G. J., Guilfoile, P., Chupp, G. L., Berman, J. S., Kim, S. J., Kornfeld, H. and Young, R. A. A chemoattractant cytokine associated with granulomas in tuberculosis and silicosis. Proc. Natl. Acad. Sci. U S A. 1997, 94, 6414-6419. Nemir, M., DeVouge, M. W. and Mukherjee, B. B. Normal rat kidney cells secrete both phosphorylated and nonphosphorylated forms of osteopontin showing different physiological properties. J. Biol. Chem. 1998, 264, 18202-18208. Pinkstaff, J. K., Detterich, J., Lynch, G. and Gall, C. Integrin subunit gene expression is regionally differentiated in adult brain. J. Neurosci. 1999, 19, 1541-1556. Shin, S. L., Cha, J. H., Chun, M. H., Chung, J. W. and Lee, M. Y. Expression of osteopontin mRNA in the adult rat brain. Neurosci. Lett. 1999, 273, 73-76. Takemoto, M., Yokote, K., Nishimura, M., Shigematsu, T., Hasegawa, T., Kon, S., Uede, T., Matsumoto, T., Saito, Y. and Mori, S. Enhanced expression of osteopontin in human diabetic artery and analysis of its functional role in accelerated atherogenesis. Arterioscler. Thromb. Vasc. Biol. 2000, 20, 624-628. Thayer, J. M. and Schoenwolf, G. C. Early expression of Osteopontin in the chick is restricted to rhombomeres 5 and 6 and to a subpopulation of neural crest cells that arise from these segments. Anat. Rec. 1998, 250, 199-209. Wang, X., Louden, C., Yue, T. L., Ellison, J. A., Barone, F. C., Solleveld, H. A. and Feuerstein, G. Z. Delayed expression of osteopontin after focal stroke in the rat. J. Neurosci. 1998, 18, 2075-2083..

(13)