대한내분비학회지 : 제 20 권제 6 호 2005 지상강좌 뼈의재형성및무기질화 서울대학교의과대학내과학교실 신찬수 조화영 Bone Remodeling and Mineralization Chan Soo Shin, Hwa Young Cho Department of Internal Medicine, Seoul National University College of Medicine, Seoul National University Hospital, Seoul, Korea 서론뼈는매우역동적인조직중의하나로물리적인지지기능과중요장기의보호기능뿐만이아니라무기물의항상성조절에중요한역할을담당하고있다. 뼈는다른결체조직과는다르게무기질화 (mineralization) 과정을거치게되고, 일생에걸쳐끊임없이재형성 (remodeling) 과정을거치게된다. 이러한무기질화와재형성과정에대한기전에대해서는많은연구들이진행되고있으며아직풀리지않은의문점들도많다. 이러한기전에대한연구는골다공증을비롯한골대사질환의병태생리와치료에있어큰진전을이루게할것이다. 본논문에서는뼈의무기질화와재형성에대한개념을이해하고중요성을살펴보고자한다. 1. 뼈의무기질화 (Bone Mineralization) 1) 세포외기질 (Extracellular matirx) 뼈의형성은간엽줄기세포 (mesenchymal stem cell) 로부터분화단계를거쳐형성된조골세포 (osteoblast) 로부터시작된다. 이러한분화과정에서 osteoblastic lineage cell은골기질을이루는수많은골기질단백질들의영향을받게되고, 이러한골기질단백질들은분화과정의단계마다특이적으로발현이된다. 이러한단백질과이를형성하는유전자에대한이해는뼈의형성과정을이해하는데있어큰역할을할것이다. 먼저골기질을이루는근간이되는세포외기질을구성하는단백질들에대해알아보도록하겠다. 가. 콜라겐 (Collagen) 골기질단백질에있어가장근간이되는단백질은제1형콜라겐 (type I collagen) 으로전체골기질의 90% 를이루고있다 (Table 1). 제1형콜라겐은 triple-helical molecule로서 2개의 α Table 1. Characteristics of Collagen-Related Genes and Proteins found in Bone matrix (from ref. 42) protein/gene Function Disease/animal model Type I-17q21.23, 7q21.3-22 [α1(i)2α2(i)] [α1(i)3] Type X-6q21-22.3 [α1(i)3] Type III-2q24.3-31 [α1(iii)3] Type V-9q34.2-34.3; 2q24.3-31, 9q34.2-34.3 [α1(v)2α2(i)] [α1(v)2α2(v)α3(v)] Most abundant protein in bone matrix(90% of organic matrix), serve as scaffolding, binds and orients other proteins that nucleate hydroxyapatite deposition Present in hypertrophic cartilage but dose not appear to regulate matrix mineralization Present in bone in trace amounts, may regulate collagen fibril diameter, their paucity in bone may explain the large diameter size of bone collagen fibrils osteogenesis imperfecta (m); oim mouse (m); mov 14 mouse (m); knock-in mouse; bones mechanically weak; mineral crystals small; some mineral outside collagen Human mutation - Schmid metaphyseal chondrodysplasia knockout mouse-no apparent skeletal phenotype Human mutation-different forms of Ehlers-Danlos syndrome - 543 -
- 대한내분비학회지 : 제 20 권제 6 호 2005 - Table 2. Gene and Proteins Characteristics of Serum proteins found in Bone matrix (from ref. 42) protein/gene function Disease/animal model Albumin-2q11-13 69-kDa, non-glycosylated, one sulfhydryl, 17 disulfide bonds, high affinity hydophobic binding pocket α2-hs glycoprotein-3q27-29 precursor protein of, cleaved to form A and B chains that are disulfide linked, Ala -Ala and Pro-Pro repeat sequences, N-linked oligosaccharides, cystatin-like domains Inhibits hydroxyapatite crystal growth Promotes endocytosis, has opsonic properties, chemoattractant for monocytic cells, bovine analog (fetuin) is a growth factor Knockout mouse-adult ectopic calcification Table 3. Gene and Proteins Characteristics of Glycoproteins in bone matrix (from ref. 42) Glycoproteins Function Disease/animal models Alkaline phosphatase (bone-liver-kidney isozyme) -1p34-36.1 two identical submits of ~80kDa, disulfide bonded, tissue specific post translational modification Osteonectin-5q31-33 ~35-45kDa, intramolecular disulfide bonds, α helical amino terminus with multiple low affinity Ca++ binding site. Ovomucoid homology, glycosylated, phosphorylated, tissue specific modifications Tetranectin-3p22-p21.3 21kDa protein composed of four identical subunits of 5.8 kda, sequence homologies with asialoprotein receptor and G3 domain of aggrecan Tenascin-C-9q33 Hexameric structure, six identical chains of 320kDA, Cys rich, EGF-like repeats, FN type III repeats potential Ca++ carrier, hydrolyzes inhibitors of mineral deposition such as pyrophosphates May mediate deposition of hydroxyapatitie, binds to growth factors, may influence cell cycle, positive regulator of bone formation Binds to plasminogen, may regulate matrix mineralization interferes with cell-fn interactions Hypophosphatasia (decreased activity), TNAP knockout mouse-growth impaired; decreased mineralization knockout mouse-decreased trabecular connectivity; decreased mineral content; increased crystal size Knockout mouse-no long bone phenotype, spinal deformity, increased mineralization in implant model Knockout mouse-no apparent skeletal phenotype 1(I) chain과 1개의 α2(i) chain으로이루어져있으며뼈뿐만이아니라인대, 상아질 (dentin), 눈의공막 (sclera), 각막 (cornea), 폐, 피부조직에서도발견이되며, 이들은모두동일하지않고 posttranslational modification 정도에차이를보이게된다. 콜라겐 α chain은 Gly-X-Y 반복형태를띠는데, 여기서 X 는대개 proline이고 Y는대개 hydroxyproline이다. 뼈에서가장두드러진 glycosylation형태는 galactosyl-hydroxylylysine 이며반면에다른연부조직에서는 glucosyl-galactosyl hydroxylysine 이다 [1,2]. 또한뼈에서보이는 cross-linking 형태또한연부조직과는다르다. 뼈에서의 cross-linking은 hydrosyallysine pathway을통해 lysyl-pyridinoline cross-link를형성하게되고, 이에반해연부조직에서는 allysine pathway를통해 hydroxylysyl-pyridinoline을형성하게된다. 뼈에서유래된제1형콜라겐 cross-links를소변에서측정하는것은골흡수 (bone resorption) 의좋은지표로알려져있다 [3]. 골기질과관련된콜라겐과이에관련된유전자를 Table 1에간략히정리하였다. 나. 비콜라겐단백질 (Noncollagenous Proteins, NCPs) NCPs는뼈단백의 10-15% 를차지하고있으며b 이들의 1/4 은외부로부터유입된다 [4]. 이중많은부분을차지하고있는것이혈장에서유래된단백질로서 α2-hs-glycoprotein과 albumin이다. 이들은산성 (acidic) 을띄고있으며 hydroxyapatite에대한친화력이있어골기질에붙게된다. 이들은뼈의무기질화에관여하며, 특히 α2-hs-glycoprotein은뼈세포의분화의조절에관여한다고알려져있다 [2]. 이에대한간략한내용은 Table 2에정리하였다. 골형성세포들은많은물질들을분비하고이는일반적으로 4개의그룹으로분류할수있다. (1) Proteoglycans, (2) Glycoproteins, (3) Glycoproteins with potential cellattachment activities, (4) γ-carboxylated (gla) proteins. 이들의정확한역할은아직잘알려져있지않지만, 무기질침착과조골세포, 파골세포의대사에영향을미칠것으로생각된다. 이에대하여 Table 3-Table 7에정리하였다. - 544 -
- 신찬수, 조화영 : 뼈의재형성및무기질화 - Table 4. Gene and Proteins Characteristics of Glycosaminoglycan-Containing Molecules, Leucine Rich Repeat Proteins(LPRs) and Hyaluronan (from ref. 42) Protein/gene Function Disease/animal model Aggrecan-15q26.1 ~2.5ⅹ10 6 intact protein, ~180-370,000 core ~100 CS chains of 25 kda, and some KS chains of similar size, G1 G2 and G3 globular domains with hyaluronan binding sites, EGF and CRP-like sequences Versican (PG-100) 5112-14 ~1ⅹ10 6 intact protein, ~360 kda core, ~12 CS chains of 45kDa, G1 and G3 globular domains with hyaluronan binding sites, EGF and CRP-like sequences Decorin (Class 1 LRR)-12q21-23 ~130 kda intact protein, ~38-45 kda core with 10 leucine rich repeat sequences, 1 CS chain of 40 kda Biglycan (Class 1 LRR)-Xq27 ~270 kda intact protein, ~38-45 kda core with 12 leucine rich repeat sequences, 2 CS chain of 40 kda Asporin (Class 1 LRR)-9q22 67 kda, most likely not GAG chains Fibromodulin (Class 2 LRR)-1q32 59 kda intact protein, 42 kda core protein, one N-linked KS chain Osteoadherin (Class 2 LRR) 85 kda intact protein, 47 kda core protein, RGD sequence Lumican (Class 2 LRR)-12q21.3-q22 70-80kDa intact protein, 37kDa core protein Osteoglycin/Mimecan (Class 3 LRR)-9q22 299 aa precursor, 105 aa mature protein, no GAG in bone, keratin sulfate in other tissues Hyaluronan-multi-gene complex Multiple proteins associated outside of the cell, structure unknown Matrix organization, retention of water and ions, resilience to mechanical forces May capture space that is destined to become bone Binds to collagen and may regulate fibril diameter, binds to TGF-β and may modulate activity, inhibits cell attachment to fibronectin May bind to collagen, may bind to TGF-β, peri-cellular environment, a genetic determinant of peak bone mass Binds to collagen, may regulate fibril formation, binds to TGF-β May mediate cell attachment Binds to collagen, may regulate fibril formation Binds to TGF-β May work with versican molecule to capture space destined to become bone Brachymorphic mouse (mutation), Accelerated growth plate calcification, nanomelic chick (mutation) abnormal bone shape Knockout-no apparent skeletal phenotype, DCN/BGN double knockout-progeroid form of Ehler s-danlos syndrome Knockout mouse, Turner s syndromeosteopenia; thin bones, decreased mineral content, increased crystal size; short stature, thin bones; Kleinfelder s disease-excessive height Fibromodulin/biglycan double knockout-joint laxity and formation of supernumery sesmoid bones Lumican/fibromodulin double knockout mouse-ectopic calcification Table 5. Gene and Proteins Characteristics of SIBLINGs (Small Integrin-Binding Ligands, N-Glycosylated Proteins) (from ref. 42) Protein/gene Function Disease/animal models Binds to cells, may regulate Knockout mouse-decreased crystal mieralization, may regulate size; increased mineral content; not proliferation, inhibits nitric oxide subject to osteoclast remodeling synthase, may regulate resistance to viral infection Osteopoontin-4q21 ~44-75 kda, polyaspartyl stretches, no disulfide boneds, glycosylated, phosphorylated, RGD located 2/3 from the N-terminal Bone Sialoprotein-4q21 ~46-75kDa, polyglutamyl stretches, no disulfide bonds, 50% carbohydrat, tyrosine-sulfated, RGD near the C terminus DMP-1 4q21 513 aminoacids predicted; acidic, RGD 2/3 from N-terminus Other SIBLINGs at 4q21-enamelin, MEPE, Dentin Sialophosphoprotein Binds to cells, may initiate mineralization Possible cell attachment protein Possible cell attachment proteins Knockout mouse-no published data on phenotype Knockout mouse-craniofacial and growth plate abnormalities MEPE-a candidate phophatonin Involved in tumor-induced osteomalacia 2. 뼈의무기질화기전뼈의무기질화는세포혹은세포외기질에칼슘, 무기질이침착되는현상을말한다. 콜라겐섬유 (fiber) 가구성하는망사구조에칼슘과인이침착하고물과혼합되어 hydroxyapatite [Ca 10(PO 4) 6(OH) 2] 라는단단한무기물질을형성하게된다. Geologic hydroxyapatite crystal과는다르게 bone mineral crystal은매우작고 ( 최대직경 200A ) 많은불순물 (carbonate, magnesium, acid phosphate) 을포함하고있다. 이는보다가용성 (soluble) 이높으며뼈가칼슘, 인, 마그네슘의저장소로서의역할을수행하도록한다 [5]. 조골세포의무기질화기전은여러가지 in vitro 및 in vivo 실험모델을통한많은연구에도불구하고잘알려져있지않다. 콜라겐이대부분을차지하는뼈의기질단백들은뼈의 - 545 -
- 대한내분비학회지 : 제 20 권제 6 호 2005 - Table 6. Gene and Proteins Characteristics of Other RGD-Containing Gycoproteins (from ref. 42) Protein/gene Function Disease/animal models Thrombospondins(1-4, COMP)-15Q-1, 6q27, 1q21-24,5q13,19p13.1 ~450 kda molecule, three identical disulfide liked subunits of ~150-180kDa, Homologies to fibrinogen, properdin, EGF, collagen, von Willebrand, P. falciparum and calmodulin, RGD at the C terminal globular domain Fibronectin-2q34 ~400kDa with 2 non-identical subunits of ~200kDa, composed of type I, II, and III repeats, RGD in the 11 th type III repeat 2/3 from N termi Vitronectin-17q11 ~70 kda, RGD close to N terminus, homology to somatomedin B, rich in cysteines, sulfated, phosphorulated Fibrillin 1 and 2-15q21.1, 5q23-q31 350 kda, EGFlike domains, RGD, cysteine motifs Cell attachment (but usually not spreading), binds to heparin, platelets, types I and V collagens, thrombin, fibrinogen, laminin, plasminogen and plasminogen activator inhibitor, histamine rich glycoprotein Binds to cells, fibrin heparin, gelatin, collagen Cell attachment protein, binds to collagen, plasminogen and plasminogen activator inhibitor, and to heparin May regulate elastic fiber formation TSP-2 knockout mouse-large colagen fibrils, thickened bones, spinal deformities Knockout mouse-lethal prior to skeletal development Fibrillin 1 mutations-marfan's syndrome, Fibrillin 2 mutationscongenital contractural arachnodactyly Table 7. Gene and Proteins Characteristics of Gamma-Carboxy Glutamic Acid-Contaning Proteins in Bone Matrix (from ref. 42) Protein/gene Function Disease/animal model Matrix Gla protein-12p ~15 kda, five gla residues, one disulfide bridge, phosphoserine residues Osteocalcin-1q25-31 ~5kDa, one disulfide bridge gla residues located in α helical region Protein S-3p11-q11.2 ~72kDa May function in cartilage metabolism, a negative regulator of mineralization May regulate activity of osteoclasts and their precursors, may mark the turning point between bone formation and resorption Primarily a liver product, but may be made by osteogenic cells Knockout mouse-excessive cartilage calcification, tiptoe walking Yoshimura mouse (mutation)- osteochondral lesions Knockout mouse, osteopetrotic mouse(mutation)-thickened bones, decreased crystal size, increased mineral content Deficiency in human-osteopenia 탄성과유연성을유지시키며다른한편으로구조적으로지지역할을한다. 콜라겐단백과이와연결되어있는비콜라겐단백들은무기질화과정과골의재형성에모두영향을미치게된다. 골의형성, 수복 (repair), 재형성에관여하는세포들역시호르몬, 기계적자극및기타외부신호에반응하여고유의역할을수행한다. 이러한세포들의세포막에존재하는지질 (lipid) 은이온의소통을조절할뿐만아니라무기질화에직접적으로관여한다. 세포내와세포외간질에존재하는수분은조직특성및영양상태의유지에중요하다. Anderson 등은골의무기질화가 extra-cellular matrix vesicle 이라는특수한기질소포에서시작된다고주장하였다 [6]. 이 vesicle은연골세포와조골세포의세포막에연결된 membrane-bound extracellular bodies로서이부위에칼슘과인을고농도로축적하게된다. 뿐만아니라이 vesicle 내에는무기질화를억제하는것으로알려진인자들 (ATP, pyrophos- phate, proteoglycan) 을제거하는효소들을함유하고있다. 또한이내부에는단백질, 산성인지질 (acidic phospholi- pid), 칼슘, 무기인등으로이루어진소위 nucleational core 를지니고있어서 apatite 형성을촉진하게된다 [2]. 그러나이 vesicle은콜라겐섬유와직접연결되어있지는않기때문에어떻게 mineral crystal이이 vesicle로부터콜라겐기질사이로이동하는지에대해서는알려져있지않다. 골간질을구성하고있는콜라겐섬유사이에 mineral crystal이침착되어무기질화가일어나기시작하는곳을 hole zone 이라고한다 [5]. 이부위는콜라겐분자가섬유를이룰때서로어긋나게배치되면서생기는분자사이의 hole에칼슘과인이최초로침착하게된다는것이다. 전술한바와같이 extracellular matrix vesicle 과이 hole zone 에서각각무기질화가시작되는지, 아니면 extracellular matrix vesicle" 에서형성된 mineral crystal이 hole zone 으로이동하는지는확실치않은상태이다. - 546 -
- 신찬수, 조화영 : 뼈의재형성및무기질화 - Table 8. Effects of Bone Matrix Proteins on Mineralization in VITRO (from ref. 42) Promote or support apatite formation Inhibits mineralization Dual function (nucleate and inhibit) No known effect on mineralization Type I collagen Proteolipid(matrix vesicle nucleational core) Aggrecan α2-hs glycoprotein Matrix gla protein(mgp) Osteopontin Osteocalcin Biglycan Osteonectin Fibronectin Bone sialoprotein Decorin BAG-75 Lumican Tetranectin Osteoaherin Thrombospondin Fig. 1. Bone remodeling is accomplished by cycles involving the resorption of old bone by osteoclasts and the subsequent formation of new bone by osteoblasts. 최초의 cystal이형성된후이온들이부착하면서첫번째안정화된결정 (critical nucleus) 을형성하게되는데, 이과정이결정화과정에있어가장에너지가많이요구되는과정이다. 이후이온들이더부착하면서결정은성장하게된다. 크리스탈의크기가증가하는것은크리스탈에새로운이온이더침착한결과일수도있고 (crystal growth) 크리스탈의응집에의해서생길수도있다. 이러한과정에있어골기질에존재하는비콜라겐단백질들이결정표면에부착하여결정의크기와모양을조절하게된다. 콜라겐은 bone mineral nucleator로알려져있고, 이후의연구들에서 bone matrix에서비콜라겐단백질을제거한경우에도결정형성에장애가발생하는것이알려졌다 [7]. 여러연구에서알려진무기질화에영향을미치는 bone matrix protein에대하여는앞서 Table 1-Table 7에언급하였다. 무기질화가되지않은뼈에서 phosphate protein을제거하는경우그정도에비례하여핵화 (nucleational) 능력이감소됨이발견되었고, 이는 bone mineral nucleators의하나가 phosphoprotein이라는점을시사한다. 뼈에존재하는 phosphoproitein에는콜라겐뿐만아니라소위 SIBLING (osteopontin, bone sialoprotein [BSP], matrix extracellular phosphoglycoprotein [MEPE], dentin matrix protein-1 [DMP-1], osteonectin, bone acidic glycoprotein-75 [BAG-75]) 이라고불리는일련의단백이있 으나 (Table 1-Table 7)[2], 현재까지 solution 상태에서 apatite nucleator로서작용하는것으로밝혀진것은 BSP뿐이며 [8], DMP-1의경우세포배양시과발현시켰을경우무기질화를가속화시킴이보고된바있다 [8,9]. Cell-free solution 또는세포배양을통해알려진무기질화에관여하는골기질단백질에대하여 Table 8에언급하였다. 2. 뼈의재형성 (Bone Remodeling) 뼈는지속적인재형성과정을거치면서유지된다. 파골세포에의해뼈가흡수, 제거되고이후조골세포에의해연속적으로새로운뼈가형성되는데, 이런두가지의연속적으로밀접한현상을 coupling event라한다 (Fig. 1). 이러한골재형성과정은뼈전체에걸쳐산재된 basic multicellular unit (BMU) 를기본으로일어나며, 이골재형성단위들은 PTH와 1,25(OH) 2D 3 등의전신적호르몬에의한영향뿐만아니라주변골의미세환경에의해국소적인조절을받게되며, 골재형성이독립된단위를기본으로이루어진다는점에서볼때, 국소적으로분비되는호르몬과사이토카인들이재형성과정에있어중요한역할을할것으로생각된다 [10]. 뼈미세환경에서골재형성과정에관여하는사이토카인들에대한연구결과들이축적되고있으며, 어떤사이토카인생성이적거나많아질때뼈의형성에중요한영향을미친다는결과들이보고되고있다 (Table 9). - 547 -
- 대한내분비학회지 : 제 20 권제 6 호 2005 - Table 9. Cytokines produced in bone microenvironment with major effects on Osteoblasts and Osteoclasts(from ref. 42) Osteoclastogenic cytokines RANK ligand Osteoprotegrin Macrophage-colony stimulating factor Interleukin-1 Tumor necrosis factor Interleukin-8 Vascular endothelial growth factor Interleukin-15, -16 and -17 Prostaglandins and leukotrienes Osteoblastogenic cytokines Transforming growth factor-β Bone morphogenic proteins Fig. 2. Relative proportions of cortical (compact) and trabecular (cancellous) bone in different parts of the skeleton. 1) 뼈의재형성과정사람의골격계구성골조직은피질골 (cortical bone) 과소주골 (trabecular bone) 로구성되어있다. 피질골은치밀골 (compact bone), 박층골 (lamellar bone) 이라고도불린다. 피질골의기본단위는골원 (osteon) 이라는형식으로되어있고, 이각각의골원들은세로로잘배열되어단단한조개껍질같은막구조를형성한다. 두번째타입의골조직은소주골로격자모양을가졌다고하여망상골 (cancellous bone) 이라명명한다. 대부분의골은치밀골과소주골조직두가지모두를가지며, 치밀골은단단한바깥덮개를형성하고소주골은내부구조를형성한다. 그러나이러한두가지조직의비율은골마다다르다. 골다공증관련골절부위로가장흔한곳중하나인요추골 (lumbar spine) 의경우소주골이 66% 이상을차지하고있으며, 대퇴전자간부위는 50% 의소주골과 50% 의피질골로이루어져있다. 반면에요골의경우 95% 이상이피질골로이루어져있다. 이렇듯부위별로골의구조에있어차이가있고더불어미세환경에도차이가있게된다 (Fig. 2). 골수와근접하게존재하는소주골의경우골수의여러세포들이분비하는사이토카인등의국소인자에의한조절을보다강하게받게되고반면에골수로부터멀리떨어져있는치밀골의경우국소조절보다는 PTH나 1,25(OH) 2D 3 와같은전신작용을하는호르몬의조절을더많이받게된다. 가. 피질골에서의재형성피질골은인체골격계의 85% 를차지하고있다. 골내막 (endosteal) 에서와 Haversian system내에서흡수가발생하고 - 548 -
- 신찬수, 조화영 : 뼈의재형성및무기질화 - 이는피질골의 porosity를증가시키게된다. 더불어골외막 (periosteal) 에서는골형성이일어나피질골의직경은커지게된다. 피질골소실은 40대들면서시작되고특히폐경이후첫 5-10년간빠른속도로골소실이발생하게되다가, 이후소실속도가완만하게된다. 피질골소실은골반, 손목골절의주요위험인자이고, 특히일차성부갑상선기능항진증이있는경우골흡수가더욱증가하게된다. 나. 소주골에서의재형성소주골은인체골격계의 15% 를차지하고있다. 소주골소실은피질골소실보다더이른시기부터발생하며, 폐경후골소실의가속도정도는피질골에서만큼뚜렷하지는않다 [11,12]. 소주골의소실은단순히 bone plate가얇아지는것으로인한것이기보다는골소주의천공 (perforation) 혹은분열 (fragmen- tation) 으로인한것인데, 이는피질골과는달리골수와접촉되어형성된미세환경에의한파골세포의국소조절의영향으로인한것이라생각된다. 골재형성의시작은파골세포활성화로부터시작된다. 시작기전에대해서는아직정확히알려져있지는않다. 한가지가설은뼈표면의변화를주변의면역세포나뼈세포들이인지하여파골세포에게신호를전달하여활성화시킨다는것인데, 이또한처음면역세포등이인지하는기전에대해서는알려진것이없다. 다. Coupling 골재형성과정중첫번째과정인골흡수과정은 10일정도가소요되며, 이후조골세포가이동하여부착하고증식, 분화하여뼈를형성하는과정은약 3개월정도가소요된다. 골흡수에이어골형성이발생하는 coupling event의세포학적기전이나 호르몬에의한조절기능등은역시잘알려지지않았으며연구중에있다. 국소적으로는골흡수과정에서분비되는여러사이토카인 (IGF-I/II, TGF-β) 이매개가되어조골세포활성화를유발한다는결과들 [13~15] 과 Runx2/Cbfa1 전사인자와신호전달과의관련성 [16], 또최근에는 leptin-hypothalamus로매개되는교감신경계활성이매개하는기전에대한연구결과들 [17,18] 이축적되고있다. 이러한골재형성기전에대한세포수준에서의기전연구는골다공증을비롯한여러골대사질환을이해하는데있어중요한열쇠가될것이다. 2) 골흡수에관여하는인자들가. Osteoprotetegrin (OPG)/RANKL/RANK OPG는 TNF receptor superfamily에속하며, 대부분의 TNFR superfamily와는달리 transmembrane domain이없는 soluble receptor이다 [19]. 쥐에서 recombinant OPG를주입한경우병적인상태뿐아니라생리적골흡수도억제하였고심한골석회화증 (osteopetrosis) 이유발되는것이관찰되었다 [20]. 이와는반대로 OPG-deficient mice의경우심한골다공증이유발되었다 [21,22]. 또한 thyroparathyroidectomized mice에서 PTH주입후상승된혈청칼슘농도가 OPG주입후급격하게감소한결과에서 OPG가 osteoclastogenesis뿐만이아니라성숙한파골세포의기능에도영향을미친다는점이알려졌다 [23]. 이후여러연구결과 OPG는 RANKL에대한 nonsignaling decoy receptor로 osteoclastogenesis를억제할뿐만아니라성숙파골세포에도영향을끼쳐 osteoclastmediated bone resorption의주요한조절단백질임이알려졌다. TNF ligand family에속하는 RANKL은파골세포의골흡수과정에있어중요한조절인자로알려진 RANK와 OPG의 Fig. 3. Osteoblast/stromal cell and osteoclast coupling mediated through RANKL/RANK interactions. - 549 -
- 대한내분비학회지 : 제 20 권제 6 호 2005 - ligand로 membrane-bound form과 soluble form 모두가존재한다 [24~26]. 골재형성에있어서의 RANKL의기능역시 knockout mice에서잘알려져있으며, RANKL knockout mice 에서는성숙한파골세포의형성이안되어심한골석화증이유발됨이알려지면서 RANKL이파골세포분화에절대적인인자임이알려지게되었다 [27,28]. TNFR superfamily에속하는 RANK는유일하게알려진 RANKL의 signaling receptor로 [29], RANK null mutant mice에서치아발육이전혀이루어지지않음이보여졌고, RANKL KO mice에서와마찬가지로심한골석화증이유발되었다 [30]. 이와같은연구결과들로 RANK/RANKL pathway가성숙한파골세포의활성화와파골세포의분화에필수적인신호전달을매개하는가장중요한신호절달체계임이증명되었다 (Fig. 3). 나. TGF-β TGF-β는면역세포에서뿐만아니라조골세포와골기질의골간질세포에서분비가되며, 이는조골세포와파골세포모두에작용하는골재형성에있어중요한조절인자중의하나이다. In vivo 및 in vitro 연구에서 TGF-β의뼈에대한역할은논란의여지가있긴하지만, 일반적인견해는 active TGF-β는골형성을자극하고 [31] 골흡수를억제하면서골흡수에이어발생하는골형성의 coupling과정에중요한역할을담당하는조절인자라는점이다. 예를들어골조직의미세환경에있어낮은농도의 TGF-β는파골세포의분화와활성을자극하지만골흡수과정동안골기질에서지속적으로분비되어고농도로축적이되게되면이는파골세포의활성의억제인자로서작용을하게되고, 동시에다 른골형성인자들과상호작용을통해조골세포활성을자극하여골형성을촉진하는역할을하게된다 [32]. 다. IL-1 IL-1와 β가있으며뼈에대한효과는동일한수용체를통해같은효과를보인다. 이들은활성화된단핵구세포뿐만아니라조골세포와종양세포등에서분비가되며이는강력한파골세포의자극인자로 RANKL을매개로파골세포형성과활성의모든과정에있어작용을하여골흡수를증가시켜골대사속도를증가시키게된다. 이는일부종양과류마티스관절염등과같은만성염증성질환에서관찰되는골흡수증가에관여하는것으로보인다 [33,34]. 라. Lymphotoxin 과 TNF-α 기능적으로 IL-1과유사한작용을하며, IL-1과 synergistic effect가있다. 마. M-CSF (CSF-1) CSF-1 null mice인 op/op variant osteopetrosis에서 CSF-1 생성장애가관찰되고, 이는정상적인파골세포형성장애를유발한다는점이관찰되었고, 이경우 CSF-1 투여로치료할수있다 [35]. 파골세포계열세포는 CSF-1 receptor (a receptor tyrosine kinase) 를발현하고있고, 이는 RANKL과 TGF-β와함께작용하여파골세포의골흡수과정에관여한다. 바. IFN-γ IFN-γ는다른면역세포에서생산되는사이토카인과는다 Fig. 4. Factors that modulate the differentiation and function of osteoblast and osteoclast. - 550 -
- 신찬수, 조화영 : 뼈의재형성및무기질화 - 르게파골세포의골흡수억제에관여한다. 이는파골세포전구세포에서성숙세포분화과정을억제를통한것으로보여지며, 이는 RANK의 downstream signaling pathway의 TRAF의분해유도를통한것으로생각된다. 3) 골형성에관여하는인자들파골세포의골흡수과정후파골세포는세포괴사를거치게되고, 이어서조골세포가골흡수부위로이동하여증식, 분화과정을거쳐골무기질화가일어나게되는과정을거치게된다. 조골세포가골흡수부위로이동하는것은골흡수과정에서생산되어분비된 active TGF-β을비롯한국소인자들과기질단백질인제1형콜라겐, osteocalcin 등에의해유도되게된다 [36]. 이후조골세포의증식과정을거치는데이과정에관여할것으로추정되는인자에는 TGF-β superfamily (TGF-βs I or II) 와 PDGF, IGFs-I, II, heparin-binding FGFs가있다 [37]. 다음단계인성숙조골세포로의분화단계로 IGF-I과 BMP-2가관여하는것으로보인다 [38,39] (Fig. 4). 4) Coupling process 에관여하는인자들 Coupling event에관여하는 osteotrophic factor에는 TGF-β, BMPs, IGFs-I,II, PDGF, FGFs가있다. 이들은골흡수과정에서국소적으로생산되어분비되는인자들로서골재형성과정에있어골흡수후골형성이발생하는과정에관여한다 (Fig. 5). 이중 TGF-β superfamily는 coupling event에서중요한역할을하고있는것으로보이며, rat등을이용한실험에서 periosteum또는 endosteum에국소적으로주입한경우주입부위에국속적으로골형성이촉진되는것이관찰되었다. 동시에골흡수에도영향을미치지만결과적으로 positive balance를보이게되는것이관찰되었다. BMPs, IGFs-I,II을주입하여시행한실험에서도결과적으로 bone mass가증가되는비슷한결과를얻었다. 최근 Wnt 단백이골형성에중요한역할을함이알려지고있다. 이 Wnt 단백들은 frizzled 단백과 LDL-receptor-related protein 5/6(LRP5/6) 에결합하여신호전달을하며 LRP5의활성화돌연변이는골량을증가시키며 [40], 반대로불활성화돌연변이는골다공증을유발함이보고되었다 [41]. Fig. 5. Growth factor concept of coupling. Fig. 6. Effects of antiresorptive agent on remodeling space and mineralization in the basic multicellular units. - 551 -
- 대한내분비학회지 : 제 20 권제 6 호 2005 - 또한지방세포에서분비되는호르몬인 leptin이골량을조절함을보고하여흥미를끌고있다. 이연구자들은조골세포에는 leptin에대한수용체가없으나, leptin 자체혹은 leptin수용체의돌연변이를지닌 ob/ob mice와 db/db mice에서골량이증가함을보고하며, leptin에의한골량의조절이시상하부를거쳐교감신경계를통해이루어진다고보고하였다 [17,18]. 이러한연구결과는매우중요한발견이나아직까지모든연구자들이이가설에동의하고있지는않다. 5) 골재형성의증가와골다공증골재형성은 basic multicellular unit (BMU) 라는단위에서이루어지며예를들어폐경이후와같은조건에서는이각각의 BMU에서골의흡수가골의형성보다클때, 이러한음의균형이발생한다. 그러나매재형성과정마다골표면에서소실되는골량은아주미세하기때문에이러한양적인불균형은골소실의일부만을설명할뿐이며더문제가되는것은이러한재형성의속도 (remodeling rate) 이다. 즉, 전신에걸쳐서 BMU가빠른속도로생기면소주골이나피질골이가늘어지고소공이많아지게되어 (porous), 전신적인골량의감소로이어진다는것이다. 현재골다공증치료제로이용되는대부분의골흡수억제제는바로이러한 BMU의생성속도, 즉 activation frequency 를현저하게낮추어골량을유지시키고골절을예방하는효과를나타내게된다 (Fig. 6). 던공간을채워가며뼈의무기질화를이루게된다. 이러한과정을 1차무기질화 (primary mineralization) 라고하며, 이과정은매우빨리진행되지만길게는 2~3개월까지도지속된다. 이러한 1차무기질화이후침착된무기질은그크기혹은수의증가에의해더욱더축적되어이를 2차무기질화 (secondary mineralization) 이라고하나, 이시기에는시간에따른무기질침착의정도가지수함수적으로감소하게된다 (Fig. 7). 무기질침착은뼈조직내에있는수분을제거하면서일어나기때문에침착되는무기질의양에는한계가있게된다. 결론이상골의무기질화및재형성과정에관여하는인자들과이들의작용에의해어떻게이두가지과정이조절되고있는지알아보았다. 아직까지도이에대한연구가끊임없이이어지고있으며, 특히최근들어인간및동물유전자에대한정보가늘어나고새로운분자생물학적기법의발달로괄목할만한성장을이루었으나, 생채내에서는골수라는접근이어려운공간에서이루어진다는면과병적인변화와생리적변화와의경계가불분명하다는점에서연구에어려운점이있다. 그러나이두가지생리적인현상에대한연구는골다공증을비롯한다양한골대사질환에대한병태생리를이해하고치료에중요한단서를제공할분야로향후에도지속적인발전이기대되고있다. 6) 1차및 2차무기질화파골세포에의해골이흡수되고나면이에 coupling되어골의형성단계가이어지는데, 먼저조골세포에의해 osteoid가합성이되고콜라겐분자사이에작은 mineral crystal이침착하게되며이후수일에걸쳐이러한 crystal이수분이차지하고있 참고문헌 1. Rossert J, Crombrugghe B: Type I collagen: Structure, synthesis and regulation. In:J. Bilezikian, L. Raisz, and G. Rodan (eds.), Principles of bone biology 1. San Diego, Fig. 7. Primary and secondary mineralization phase. The secondary mineralization period is defined as the time required for bone to mineralize from 70% to 95% of the theoretical maximum ash fraction. - 552 -
- 신찬수, 조화영 : 뼈의재형성및무기질화 - USA: Academic Press, 2002 2. Gokhale J, Robey P, Boskey A: The biochemistry of bone. In: R. Marcus, D. Feldman, and A. Kelsey (eds.), Osteoporosis 1, pp107-188, San Diego, USA: Academic Press, 2001 3. Seibel MJ, Woitge HW: Basic principles and clinical applications of biochemical markers of bone metabolism: biochemical and technical aspects. J Clin Densitom 2:299-321, 1999 4. Gorski JP: Is all bone the same? Distinctive distributions and properties of non-collagenous matrix proteins in lamellar vs. woven bone imply the existence of different underlying osteogenic mechanisms. Crit Rev Oral Biol Med 9:201-223, 1998 5. Glimcher MJ: Mechanism of calcification: role of collagen fibrils and collagen-phosphoprotein complexes in vitro and in vivo. Anat Rec 224:139-153, 1989 6. Anderson HC: Mineralization by matrix vesicles. Scan Electron Microsc 953-964, 1984 7. Termine JD, Belcourt AB, Conn KM, Kleinman HK: Mineral and collagen-binding proteins of fetal calf bone. J Biol Chem 256:10403-10408, 1981 8. Stubbs JT, 3rd, Mintz KP, Eanes ED, Torchia DA, Fisher LW: Characterization of native and recombinant bone sialoprotein: delineation of the mineral-binding and cell adhesion domains and structural analysis of the RGD domain. J Bone Miner Res 12:1210-1222, 1997 9. Boskey AL: Osteopontin and related phosphorylated sialoproteins: effects on mineralization. Ann N Y Acad Sci 760:249-256, 1995 10. Frost H. Dynamics of bone remodeling. Bone Biodynamics, pp315. Boston, MA: Little and Brown, 1964. 11. Riggs BL, Wahner HW, Melton LJ, 3rd, Richelson LS, Judd HL, Offord KP: Rates of bone loss in the appendicular and axial skeletons of women. Evidence of substantial vertebral bone loss before menopause. J Clin Invest 77:1487-1491, 1986 12. Genant HK, Cann CE, Ettinger B, Gordan GS: Quantitative computed tomography of vertebral spongiosa: a sensitive method for detecting early bone loss after oophorectomy. Ann Intern Med 97:699-705, 1982 13. Rodan GA, Martin TJ: Role of osteoblasts in hormonal control of bone resorption--a hypothesis. Calcif Tissue Int 33:349-351, 1981 14. Howard GA, Bottemiller BL, Turner RT, Rader JI, Baylink DJ: Parathyroid hormone stimulates bone formation and resorption in organ culture: evidence for a coupling mechanism. Proc Natl Acad Sci U S A 78:3204-3208, 1981 15. Locklin RM, Khosla S, Turner RT, Riggs BL: Mediators of the biphasic responses of bone to intermittent and continuously administered parathyroid hormone. J Cell Biochem 89:180-90, 2003 16. Enomoto H, Shiojiri S, Hoshi K, Furuichi T, Fukuyama R, Yoshida CA, Kanatani N, Nakamura R, Mizuno A, Zanma A, Yano K, Yasuda H, Higashio K, Takada K, Komori T: Induction of osteoclast differentiation by Runx2 through receptor activator of nuclear factorkappab ligand (RANKL) and osteoprotegerin regulation and partial rescue of osteoclastogenesis in Runx2-/- mice by RANKL transgene. J Biol Chem 278:23971-23977, 2003 17. Fu L, Patel MS, Bradley A, Wagner EF, Karsenty G: The molecular clock mediates leptin-regulated bone formation. Cell 122:803-815, 2005 18. Elefteriou F, Ahn JD, Takeda S, Starbuck M, Yang X, Liu X, Kondo H, Richards WG, Bannon TW, Noda M, Clement K, Vaisse C, Karsenty G: Leptin regulation of bone resorption by the sympathetic nervous system and CART. Nature 434:514-520, 2005 19. Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, Nguyen HQ, Wooden S, Bennett L, Boone T, Shimamoto G, DeRose M, Elliott R, Colombero A, Tan HL, Trail G, Sullivan J, Davy E, Bucay N, Renshaw-Gegg L, Hughes TM, Hill D, Pattison W, Campbell P, Sander S, Van G, Tarpley J, Derby P, Lee R, Boyle WJ: Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 89:309-319, 1997 20. Akatsu T, Murakami T, Ono K, Nishikawa M, Tsuda E, Mochizuki SI, Fujise N, Higashio K, Motoyoshi K, Yamamoto M, Nagata N: Osteoclastogenesis inhibitory factor exhibits hypocalcemic effects in normal mice and in hypercalcemic nude mice carrying tumors associated with humoral hypercalcemia of malignancy. Bone 23:495-498, 1998 21. Bucay N, Sarosi I, Dunstan CR, Morony S, Tarpley J, Capparelli C, Scully S, Tan HL, Xu W, Lacey DL, Boyle WJ, Simonet WS: osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev 12:1260-1268, 1998-553 -
- 대한내분비학회지 : 제 20 권제 6 호 2005-22. Mizuno A, Amizuka N, Irie K, Murakami A, Fujise N, Kanno T, Sato Y, Nakagawa N, Yasuda H, Mochizuki S, Gomibuchi T, Yano K, Shima N, Washida N, Tsuda E, Morinaga T, Higashio K, Ozawa H: Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/ osteoprotegerin. Biochem Biophys Res Commun 247:610-615, 1998 23. Yamamoto M, Murakami T, Nishikawa M, Tsuda E, Mochizuki S, Higashio K, Akatsu T, Motoyoshi K, Nagata N: Hypocalcemic effect of osteoclastogenesis inhibitory factor/osteoprotegerin in the thyroparathyroidectomized rat. Endocrinology 139:4012-4015, 1998 24. Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A, Qian YX, Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J, Boyle WJ: Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93:165-176, 1998 25. Lum L, Wong BR, Josien R, Becherer JD, Erdjument- Bromage H, Schlondorff J, Tempst P, Choi Y, Blobel CP: Evidence for a role of a tumor necrosis factor-alpha (TNF-alpha)-converting enzyme-like protease in shedding of TRANCE, a TNF family member involved in osteoclastogenesis and dendritic cell survival. J Biol Chem 274:13613-13618, 1999 26. Wong BR, Josien R, Choi Y: TRANCE is a TNF family member that regulates dendritic cell and osteoclast function. J Leukoc Biol 65:715-724, 1999 27. Kong YY, Boyle WJ, Penninger JM: Osteoprotegerin ligand: a common link between osteoclastogenesis, lymph node formation and lymphocyte development. Immunol Cell Biol 77:188-193, 1999 28. Kong YY, Yoshida H, Sarosi I, Tan HL, Timms E, Capparelli C, Morony S, Oliveira-dos-Santos AJ, Van G, Itie A, Khoo W, Wakeham A, Dunstan CR, Lacey DL, Mak TW, Boyle WJ, Penninger JM: OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 397:315-323, 1999 29. Li J, Sarosi I, Yan XQ, Morony S, Capparelli C, Tan HL, McCabe S, Elliott R, Scully S, Van G, Kaufman S, Juan SC, Sun Y, Tarpley J, Martin L, Christensen K, McCabe J, Kostenuik P, Hsu H, Fletcher F, Dunstan CR, Lacey DL, Boyle WJ: RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. Proc Natl Acad Sci U S A 97:1566-1571, 2000 30. Dougall WC, Glaccum M, Charrier K, Rohrbach K, Brasel K, De Smedt T, Daro E, Smith J, Tometsko ME, Maliszewski CR, Armstrong A, Shen V, Bain S, Cosman D, Anderson D, Morrissey PJ, Peschon JJ, Schuh J: RANK is essential for osteoclast and lymph node development. Genes Dev 13:2412-2424, 1999 31. Noda M, Camilliere JJ: In vivo stimulation of bone formation by transforming growth factor-beta. Endocrinology 124:2991-2994, 1989 32. Filvaroff E, Erlebacher A, Ye J, Gitelman SE, Lotz J, Heillman M, Derynck R: Inhibition of TGF-beta receptor signaling in osteoblasts leads to decreased bone remodeling and increased trabecular bone mass. Development 126:4267-4279, 1999 33. Sabatini M, Boyce B, Aufdemorte T, Bonewald L, Mundy GR: Infusions of recombinant human interleukins 1 alpha and 1 beta cause hypercalcemia in normal mice. Proc Natl Acad Sci U S A 85:5235-5239, 1988 34. Pacifici R, Rifas L, McCracken R, Vered I, McMurtry C, Avioli LV, Peck WA: Ovarian steroid treatment blocks a postmenopausal increase in blood monocyte interleukin 1 release. Proc Natl Acad Sci U S A 86:2398-2402, 1989 35. Felix R, Cecchini MG, Fleisch H: Macrophage colony stimulating factor restores in vivo bone resorption in the op/op osteopetrotic mouse. Endocrinology 127:2592-2594, 1990 36. Pfeilschifter J, Bonewald L, Mundy GR: Characterization of the latent transforming growth factor beta complex in bone. J Bone Miner Res 5:49-58, 1990 37. Canalis E, McCarthy T, Centrella M: Growth factors and the regulation of bone remodeling. J Clin Invest 81:277-281, 1988 38. Canalis E, Pash J, Gabbitas B, Rydziel S, Varghese S: Growth factors regulate the synthesis of insulin-like growth factor-i in bone cell cultures. Endocrinology 133:33-38, 1993 39. Krane SM: Identifying genes that regulate bone remodeling as potential therapeutic targets. J Exp Med 201: 841-843, 2005 40. Little RD, Carulli JP, Del Mastro RG, Dupuis J, Osborne M, Folz C, Manning SP, Swain PM, Zhao SC, Eustace B, Lappe MM, Spitzer L, Zweier S, Braunschweiger K, Benchekroun Y,Hu X, Adair R, Chee L, FitzGerald MG, Tulig C, Caruso A, Tzellas N, Bawa A, Franklin B, - 554 -
- 신찬수, 조화영 : 뼈의재형성및무기질화 - McGuire S, Nogues X, Gong G, Allen KM, Anisowicz A, Morales AJ, Lomedico PT, Recker SM, Van Eerdewegh P, Recker RR, Johnson ML: A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant high-bone-mass trait. Am J Hum Genet 70:11-19, 2002 41. Gong Y, Slee RB, Fukai N, Rawadi G, Roman-Roman S, Reginato AM, Wang H, Cundy T, Glorieux FH, Lev D, Zacharin M, Oexle K, Marcelino J, Suwairi W, HeegerS, Sabatakos G, Apte S, Adkins WN, Allgrove J, Arslan-Kirchner M, Batch JA, Beighton P, Black GC, Boles RG, Boon LM, Borrone C, Brunner HG, Carle GF, Dallapiccola B, De Paepe A, Floege B, Halfhide ML, Hall B, Hennekam RC, Hirose T, Jans A, Juppner H, Kim CA, Keppler-Noreuil K, Kohlschuetter A, LaCombe D, Lambert M, Lemyre E, Letteboer T, Peltonen L, Ramesar RS, Romanengo M, Somer H, Steichen-Gersdorf E, Steinmann B, Sullivan B, Superti-Furga A, Swoboda W, van den Boogaard MJ, Van Hul W, Vikkula M, Votruba M, Zabel B, Garcia T, Baron R, Olsen BR, Warman ML: LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development. Cell 107:513-523, 2001 42. Robey PG, Boskey AL. Extracellular matrix and biomineralization of bone. In:M. J. Favus (ed.), Primer on the metabolic bone diseases and disorders of mineral metabolism, pp38-46. Washington D.C.: American Society of Bone and Mineral Research, 2003-555 -