Curr Osteoporos Rep (2014) 12:439–445 DOI 10.1007/s11914-014-0232-1
SKELETAL DEVELOPMENT (E SCHIPANI AND E ZELZER, SECTION EDITORS)
Extracellular Matrix and Developing Growth Plate Johanna Myllyharju
Published online: 12 September 2014 # Springer Science+Business Media New York 2014
Abstract Growth plate is a specialized cartilaginous structure that mediates the longitudinal growth of skeletal bones. It consists of ordered zones of chondrocytes that secrete an extracellular matrix (ECM) composed of specific types of collagens and proteoglycans. Several heritable human skeletal dysplasias are caused by mutations in these ECM components and this review focuses on the roles of type II, IX, X, and XI collagens, aggrecan, matrilins, perlecan, and cartilage oligomeric matrix protein in the growth plate as deduced from human disease phenotypes and mouse models. Substantial advances have been achieved in deciphering the interaction networks and individual roles of these components in the construction of the growth plate ECM. Furthermore, ER stress and other cellular responses have been identified as key downstream effects of the ECM mutations contributing to abnormal growth plate development. The next challenge is to utilize the molecular level knowledge for the development of potential therapeutics. Keywords Growth plate . Extracellular matrix . Collagen . Aggrecan . Matrilin . Perlecan . Cartilage oligomeric matrix protein . ER stress
Introduction The majority of skeletal bones are formed via a process called endochondral ossification where an initial cartilaginous model is replaced by bone tissue [1–5]. Longitudinal growth of bones occurs via a specialized cartilaginous structure, the growth
J. Myllyharju (*) Oulu Center for Cell-Matrix Research, Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, PO Box 5000, FIN-90014 Oulu, Finland e-mail: [email protected]
plate. It consists of ordered zones of proliferating and differentiating chondrocytes (Fig. 1) that secrete a cartilaginous extracellular matrix (ECM) that is composed of specific types of collagens and proteoglycans. Ultimately, the growth plate chondrocytes differentiate into nondividing hypertrophic chondrocytes that undergo apoptosis and are replaced by bone cells (Fig. 1). The major collagen in growth plate cartilage is the fibril-forming type II collagen, other collagen types being the type XI and IX collagens. Type XI collagen also assembles into fibrils that are typically found within the core of the type II collagen fibrils, while type IX collagen, a fibril-associated collagen, resides at the surface of the type II/XI fibrils [6, 7]. Hypertrophic chondrocytes in the growth plate are characterized by the production of type X collagen. The growth plate cartilage also consists of a number of proteoglycans, such as aggrecan as the major component, perlecan, decorin, fibromodulin and lumican [1, 8, 9]. Other noncollagenous proteins of the growth plate cartilage include, eg, matrilins, COMP, tenascin-C, and link proteins [1, 8, 9]. The importance of the growth plate cartilage ECM is evident based on the existence of a number of human skeletal dysplasias caused by mutations in its components [10]. Besides the ECM components, several transcription factors, circulating hormones, growth factors, and cell surface receptors participate in the regulation of the growth plate cartilage [1–5, 11]. This review concentrates on the roles of the ECM components in growth plate cartilage as deduced from human disease phenotypes and mouse models. The effects of ECM component mutations can manifest by various mechanisms. The absence of a particular component can either be deleterious or tolerable, a mutant form of the component can lead to abnormal interactions and assembly of the ECM or the mutant form is not properly secreted from the endoplasmic reticulum (ER) and causes ER stress.
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ECM with extremely short and curved fibrils instead of a normal dense network of long type II collagen fibrils [28].
Type XI Collagen Type XI collagen is an α1(XI)α2(XI)α3(XI) heterotrimer. The α1(XI) and α2(XI) chains are encoded by the COL11A1 and COL11A2 genes, while the α3(XI) chain is encoded by the COL2A1 gene and is, thus, essentially identical to the α1(II) chain but has certain differences in its posttranslational modifications [13–16]. Collagen XI is found only in the thin collagen II fibrils and it is thought to regulate the fibril diameter [7, 12]. Mutations in the COL11A1 and COL11A2 genes have been identified in five distinct autosomal dominant or recessive skeletal disorders, examples being Stickler syndrome type 2 and Marshall syndrome with typical arthro-ophthalmologic features [10]. A spontaneous Col11a1 mutation that causes a premature stop codon exists in mice. Homozygous mice with this mutation have completely disorganized growth plates and severe chondrodysplasia and they die at birth [31]. Fig. 1 Schematic figure of the structure of growth plate
Type II Collagen Type IX Collagen The major collagen in growth plate cartilage is type II collagen that forms fibrils with thick (40 nm) and thin (16 nm) diameters [7, 12]. Type II collagen is a homotrimer composed of three identical α1(II) collagen chains encoded by the COL2A1 gene [13–16]. Mutations in the COL2A1 gene lead to at least ten autosomal dominant chondrodysplasias of varying severity, examples ranging from lethal forms (achondrogenesis type II and hypochondrogenesis) to diseases characterized by severe (spondyloepiphyseal dysplasia, Kniest dysplasia) or mild (Stickler syndrome) skeletal abnormalities [10]. The phenotype of type II collagen null mice resembles human achondrogenesis type II [17]. The Col2a1 null mice die immediately before or at birth, they are markedly smaller than their littermates, their long bones lack endochondral bone and the epiphyseal growth plate and intervertebral discs are not developed [17, 18]. Mice with various spontaneous or transgenic mutations in the Col2a1 gene typically display growth plate anomalies and chondrodysplasia [19–30]. For example transgenic mice with a dominant negative mutation in Col2a1 corresponding to a human mutation causing spondyloepiphyseal dysplasia have been shown to be neonatally lethal [28]. The mice had short bones, cleft palate, and respiratory failure. The proliferative chondrocytes of the growth plate were not arranged into distinct columns and thus, the proliferative zone was short and disorganized and accompanied with an enlargement of the prehypertrophic zone [28]. The epiphyseal cartilage of the mutant mice contained a loose
Type IX collagen is a heterotrimer consisting of three distinct collagen chains, α1(IX)α2(IX)α3(IX), encoded by the COL9A1, COL9A2, and COL9A3 genes [13–16]. Type IX collagen is associated to the surface of the type II/XI fibrils and is likely to regulate interactions between the fibrils themselves and with other ECM components. Mutations in the COL9A1, COL9A2, and COL9A3 genes are known to cause autosomal dominant multiple epiphyseal dysplasia and autosomal recessive Stickler syndrome [10]. Transgenic mice with a large in-frame deletion in Col9a1 suffer from mild chondrodysplasia and osteoarthritis [32]. Collagen IX null mice with inactivated Col9a1 gene were initially reported to have no obvious abnormalities at birth and apparently normal collagen fibrils were present in the rib cartilage [33, 34]. However, the mice developed osteoarthritis upon aging [33]. Later, it has been shown that lack of collagen IX has serious effects on the growth plate cartilage [35]. The columnar organization of chondrocytes was lost in the proliferative and hypertrophic zones of the mutant growth plate and its central regions were hypocellular [35]. It was also shown that glycosaminoglycan and β1 integrin distribution was abnormal and proliferation of chondrocytes was decreased in the mutant mice [35]. The mutant newborn mice also had a reduced length of the diaphysis of long bones, while the width was increased [35]. Interestingly, these abnormalities were attenuated in adult mice [35].
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Type X Collagen Type X collagen is an α1(X)3 homotrimer encoded by the COL10A1 gene [13–16]. It is specifically expressed in hypertrophic chondrocytes and assembles into hexagonal networklike structures [13–16]. Mutations in COL10A1 lead to autosomal dominant Schmid metaphyseal dysplasia [10]. Transgenic mice expressing a dominant negative mutant form of chicken collagen X have skeletal and hematopoietic defects [36, 37]. Endochondral ossification was affected in the mutant mice and was manifested as variable skeletal phenotypes ranging from thoracolumbar kyphosis and lethality at about 3 weeks of age to variable degrees of dwarfism with susceptibility to skeletal deformities. The hypertrophic zone of the growth plate was compressed in all the mutant mice, the degree of compression correlating with the severity of the phenotype [36, 37]. In contrast, analysis of the first collagen X null mouse line generated showed no obvious abnormalities in the development and growth of long bones [38]. However, phenotypic changes partly resembling Schmid metaphyseal dysplasia were observed in a second independently generated collagen X null mouse line [39]. The height of the resting zone of growth plate chondrocytes was reduced and the architecture of trabecular bone was altered in the chondro-osseous junction in the collagen X null mice [39]. In addition, differences in the distribution and organization of growth plate ECM components were observed [39]. Interestingly, it was later noticed that about 11 % of the collagen X null mice from the line generated first do not survive beyond 3 weeks of age [40]. In the perinatal lethal collagen X null mice, compression of the proliferative zone of the growth plate was observed and trabecular bone was reduced [40]. Transgenic mice expressing collagen X containing a deletion equivalent to a mutation found in human Schmid metaphyseal dysplasia patients suffer from dwarfism and have short limbs [41, 42]. The mutant collagen X was found to accumulate within the hypertrophic chondrocytes in which ER stress was observed [42]. However, the hypertrophic chondrocytes were not directed to an apoptotic pathway but utilized adaptive mechanisms including changes in cell cycle regulation, reversion to a prehypertrophic state and altered terminal differentiation [42]. Knock-in mice with a collagen X Asn617Lys mutation corresponding to a human mutation found in the Schmid metaphyseal dysplasia also had short limbs [43]. The hypertrophic zone of the growth plate was expanded, and the hypertrophic chondrocytes retaining the mutant collagen X displayed ER stress and unfolded protein response [43].
Aggrecan Aggrecan is a large chondroitin sulfate proteoglycan and is the major proteoglycan component of cartilage [5, 9]. The
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chondroitin sulfate side chains of aggrecan generate a high negative charge density and create an osmotic environment that retains water, which provides cartilage tissue with high compressive endurance. Human mutations in aggrecan cause two types of spondyloepiphyseal dysplasia, the autosomal dominant Kimberley type and autosomal recessive Aggrecan type, and autosomal dominant familial osteochondritis dissecans [10]. A natural short deletion in the mouse gene encoding aggrecan that leads to a truncated protein causes an autosomal recessive cartilage matrix deficiency syndrome with abnormal craniofacial structures, short limbs and tail and perinatal lethality [44]. The organization of chondrocytes is severely disrupted, the amount of hypertrophic chondrocytes is significantly reduced and expression of other ECM genes is altered in the growth plates of the mutant mice [45].
Matrilins Matrilins are multi-domain proteins consisting of von Willebrand factor A domain(s), epidermal growth factor-like motif(s) and a C-terminal coiled-coil domain that is responsible for the homo- and hetero-oligomerization of the matrilins [46]. The matrilin family consists of matrilins 1–4, of which matrilins 1 and 3 are mainly found in cartilage, matrilins 2 and 4 being detected also in other tissues [46]. Matrilins associate with cartilage collagen fibrils and bind covalently to aggrecan and are, thus, regarded as adaptor proteins in ECM assembly [46]. Mutations in the human gene encoding matrilin-3 have been identified in autosomal dominant multiple epiphyseal dysplasia type 5 and in autosomal recessive spondyloepi metaphyseal dysplasia Matrilin type [10]. No obvious abnormalities in skeletal development and cartilage ECM were initially discovered in matrilin-1, -2, and -3 knockout mice suggesting mutual compensatory capacity [47–49]. However, despite normal skeletogenesis and growth plate structure, abnormal fibrillogenesis of type II collagen and fibril organization at the maturation zone of the growth plate was observed in matrilin-1 knockout mice [50] and premature maturation of chondrocytes to prehypertrophic and hypertrophic chondrocytes and expansion of the hypertrophic zone of the growth plate was observed in matrilin-3 knockout mice with increased bone mineral density and susceptibility to osteoarthritis [51]. Furthermore, matrilin-1/matrilin-3 double null mice have no apparent skeletal abnormalities, although certain changes were observed in the cartilage ECM including increased collagen fibril diameters and collagen volume density in the epiphysis and growth plate [52]. Interestingly, knock-in mice with a matrilin-3 mutation (Val194Asp) known to cause multiple epiphyseal dysplasia in humans, suffer from progressive dysplasia and short-limbed dwarfism [53]. The mutant matrilin-3 was found to be retained within the ER and
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associated with induction of the unfolded protein response [53, 54]. Furthermore, proliferation of the growth plate chondrocytes was reduced and apoptosis was spatially dysregulated [53]. Combination with a null matrilin-1 mutation did not enhance the skeletal abnormalities observed in the Val194Asp matrilin-3 mutant mice [55].
Perlecan Perlecan is a large heparan sulfate/chondroitin sulfate proteoglycan and is a ubiquitous component of basement membranes, articular cartilage and growth plate [56–58]. The large protein core of perlecan consists of five domains that contain a sperm-agrin-enterokinase module and repeats similar to those present in the low-density lipoprotein receptor, immunoglobulin, laminin and epidermal growth factor, ie, molecules involved in eg, lipid metabolism, cell proliferation, and adhesion [56, 57]. Perlecan interacts with a large variety of molecules including growth factors and their receptors, integrins, and various ECM components and it has been shown to be involved in eg, angiogenesis, cardiovascular development and neurogenesis [56–58]. In addition, perlecan is essential for cartilage and bone formation. Mutations in the human perlecan gene cause three autosomal recessive skeletal disorders, the Silverman-Handmaker type and RollandDesbuquois type dyssegmental dysplasias and the SchwartzJampel syndrome (myotonic chondrodystrophy) [10]. Many of the perlecan null mice are embryonic lethal around midgestation because of abnormal cephalic development or heart failure [59, 60]. The surviving embryos have progressive defects in skeletal development starting at E14.5 when endochondral ossification begins and the mice die shortly after birth [59, 60]. The zonal and columnar arrangement of chondrocytes was extremely disorganized in the growth plate and endochondral ossification was reduced, type II collagen fibrils were sparse and disorganized and the proliferation of chondrocytes was reduced [59, 60]. It has been shown that the chondroitin sulfate side chains of perlecan enhance fibril formation of type II collagen fibrils [61]. Furthermore, the absence of perlecan was also found to affect fibroblast growth factor signaling in the growth plate [59]. Knock-in perlecan mutant mice with a Cys1532Tyr mutation have been generated to model the Schwartz-Jampel syndrome [62]. However, the knock-in mice had no obvious anatomic phenotype and the mutation did not affect the transcription level, secretion, or deposition of perlecan [62]. Interestingly, when the neomycin selection cassette was retained in the Cys1532Tyr mutant mice, they displayed skeletal abnormalities closely resembling the disease phenotype [62, 63]. In these mice, transcription of the mutant perlecan gene was altered (reduced level of fulllength mutant perlecan mRNA and emergence of a truncated transcript) and secretion of perlecan was reduced [62].
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Perlecan has also been shown to regulate VEGF signaling and to be essential for vascularization during endochondral bone formation [64].
Cartilage Oligomeric Matrix Protein Cartilage oligomeric matrix protein (COMP, thrombospondin 5) is a pentameric protein composed of five identical subunits that are linked via their N-terminal coiled-coil domain, which is followed by EGF-like and thrombospondin type 3 repeats and a C-terminal globular domain that can interact with several other ECM proteins including type I, II, and IX collagens, matrilins and fibronectin [4, 9, 65]. Mutations in the human gene coding for COMP cause autosomal dominant pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia type 1 [10]. The COMP mutations act in a dominant negative manner and cause retention of the mutant COMP polypeptides in the ER leading to unfolded protein response, cell death, and reduction of chondrocytes in the growth plate [65]. In addition to intracellular retention of COMP, studies with PSACH mutant chondrocytes showed that the COMP mutations simultaneously reduced secretion of type IX collagen and matrilin-3, but not that of type II collagen or aggrecan, leading to marked reduction in collagen IX and matrilin-3 in the matrix [66]. As in the case of matrilin-3 null mice, COMP null mice seem to have normal skeletal development [67] and simultaneous deficiency of COMP did not aggravate the phenotype of collagen IX null mice [68]. In contrast, mice harboring PSACH mutations in COMP manifest several of the disease characteristics [65]. Transgenic mice expressing a COMP mutant with a deletion of Asp469 showed slight growth retardation in the males, abnormal columnar organization of chondrocytes in the growth plate with atypical collagen fibrils and loss of proteoglycans and ER stress [69]. Although PSACH is an autosomal dominant disorder, heterozygous mice with a knock-in Asp469del COMP mutation showed no obvious skeletal defects, but the homozygous Asp469del mutant mice had progressive short-limb dwarfism and disorganized chondrocyte columns in the growth plate with hypocellular areas in the resting and upper proliferative zones [70]. The mutant COMP was retained within the ER together with matrilin-3 and to some extent with collagen IX with concomitant reduction in their extracellular deposition and ultrastructural abnormalities of the ECM [70]. Interestingly, although dilation of the ER was detected, unfolded protein response was not induced but instead changes in the expression of genes involved in oxidative stress, apoptosis, and cell cycle arrest were observed [70]. Heterozygous knockin mice with a COMP Thr583Met mutation causing mild PSACH or multiple epiphyseal dysplasia type 1 also had no significant changes in their skeletogenesis, but the homozygous mice developed short-limb dwarfism by 9 weeks of age
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[71]. The chondrocytes in the growth plate were sparse, their columnar organization was disturbed from 2 weeks of age and their proliferation was decreased and apoptosis was increased [71]. Unfolded protein response was detected in the chondrocytes, but significant amounts of the mutant COMP were still secreted into the growth plate ECM, which showed ultrastructural abnormalities and changes in the localization of the interacting proteins matrilin-3 and collagen IX [71]. In all these mouse lines with disease-causing COMP mutations the phenotype has been milder than in the corresponding human patients. A mouse line that more closely models the human PSACH phenotype at the cellular and growth plate level has been generated using a tetracycline-inducible transgene approach and used to analyze the disease causing mechanisms in detail at cellular level [72–74].
ECM Mutations and ER Stress As indicated above ER stress with or without induction of the unfolded protein response is commonly observed in growth plate chondrocytes expressing mutant ECM components and is typically associated with changes in chondrocyte proliferation and apoptosis [75]. Similarly, it has been shown that chondrocytes with reduced amount of collagen prolyl 4-hydroxylase, an essential enzyme of collagen synthesis [13], leads to accumulation of ECM components within the cells and ER stress [76]. To study the contribution of chondrocyte ER stress to abnormal skeletogenesis independent of any ECM mutations, a transgenic mouse line expressing mutant thyroglobulin specifically in the chondrocytes has been generated [77••]. The mutant thyroglobulin is known to cause ER stress in thyrocytes and was found to cause short-limb dwarfism in the transgenic mice [77••]. The ECM of the growth plates of the transgenic mice was apparently normal, but the chondrocyte columns were disordered with hypocellular areas [77••]. Chondrocyte proliferation was reduced and showed moderate cellular stress without induction of the unfolded protein response or apoptosis [77••]. These data show that ER stress is sufficient to cause abnormal skeletogenesis. ER stress can be alleviated chemically by 4-sodium phenylbutyrate and its effect has been studied on mice expressing the Val194Asp mutant matrilin-3 or the Asn617Lys mutant collagen X [54]. However, no increases in the secretion of the mutant proteins or improvement of the disease phenotype were observed, the lack of efficacy being potentially because of the avascularity of the growth plate cartilage [54].
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critical roles in the survival, differentiation, and function of growth plate chondrocytes and thus, normal skeletogenesis. Significant progress has been made in understanding the interaction networks of the ECM components and their individual roles in the assembly of the growth plate ECM. Furthermore, ER stress and other cellular responses have been identified as key downstream effects of the ECM mutations contributing to abnormal growth plate development. The challenge in the future is to utilize the current molecular level knowledge for the development of potential therapeutics. Compliance with Ethics Guidelines Conflict of Interest J. Myllyharju has received research grants from FibroGen Inc. Human and Animal Rights and Informed Consent All studies by the authors involving animal and/or human subjects were performed after approval by the appropriate institutional review boards. When required, written informed consent was obtained from all participants.
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Conclusions Based on evidence from human heritable diseases and genemodified animal models, certain ECM components have
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445 vascularization in endochondral bone formation. Matrix Biol. 2012;31:234–45. 65. Posey KL, Alcorn JL, Hecth JT. Pseudoachondroplasia/COMP— translating from the bench to the bedside. Matrix Biol. 2014. doi:10. 1016/j.matbio.2014.05.006. 66. Hecht JT, Hayes E, Haynes R, Cole WG. COMP mutations, chondrocyte function and cartilage matrix. Matrix Biol. 2005;23:525– 33. 67. Svensson L, Aszódi A, Heinegård D, Hunziker EB, Reinholt FP, Fässler R, et al. Cartilage oligomeric matrix protein-deficient mice have normal skeletal development. Mol Cell Biol. 2002;22:4366–71. 68. Blumbach K, Niehoff A, Paulsson M, Zaucke F. Ablation of collagen IX and COMP disrupts epiphyseal cartilage architecture. Matrix Biol. 2008;27:306–18. 69. Schmitz M, Niehoff A, Miosge N, Smyth N, Paulsson M, Zaucke F. Transgenic mice expressing D469Δ mutated cartilage oligomeric matrix protein (COMP) show growth plate abnormalities and sternal malformations. Matrix Biol. 2008;27:67–85. 70. Suleman F, Gualeni B, Gregson HJ, Leighton MP, Piróg KA, Edwards S, et al. A novel form of chondrocyte stress is triggered by a COMP mutation causing pseudoachondroplasia. Hum Mutat. 2012;33:218–31. 71. Piróg-Garcia KA, Meadows RS, Knowles L, Heinegård D, Thornton DJ, Kadler KE, et al. Reduced cell proliferation and increased apoptosis are significant pathological mechanisms in a murine model of mild pseudoachondroplasia resulting from a mutation in the C-terminal domain of COMP. Hum Mol Genet. 2007;16:2072–88. 72. Posey KL, Veerisetty AC, Liu P, Wang HR, Poindexter BJ, Bick R, et al. An inducible cartilage oligomeric matrix protein mouse model recapitulates human pseudoachondroplasia phenotype. Am J Pathol. 2009;175:1555–63. 73. Posey KL, Coustry F, Veerisetty AC, Liu P, Alcorn JL, Hecht JT. Chop (Ddit3) is essential for D469del-COMP retention and cell death in chondrocytes in an inducible transgenic mouse model of pseudoachondroplasia. Am J Pathol. 2012;180:727–37. 74. Posey KL, Coustry F, Veerisetty AC, Liu P, Alcorn JL, Hecht JT. Chondrocyte-specific pathology during skeletal growth and therapeutics in a murine model of pseudoachondroplasia. J Bone Miner Res. 2014;29:1258–68. 75. Tsang KY, Chan D, Bateman JF, Cheah KS. In vivo cellular adaptation to ER stress: survival strategies with double-edged consequences. J Cell Sci. 2010;123:2145–54. 76. Bentovim L, Amarilio R, Zelzer E. HIF1α is a central regulator of collagen hydroxylation and secretion under hypoxia during bone development. Development. 2012;139:4473–83. 77.•• Gualeni B, Rajpar MH, Kellogg A, Bell PA, Arvan P, BootHandford RP, et al. A novel transgenic mouse model of growth plate dysplasia reveals the decreased chondrocyte proliferation due to chronic ER stress is a key factor in reduced bone growth. Dis Model Mech. 2013;6:1414–25. The report shows that ER stress independent of ECM mutations is a key contributor in abnormal skeletogenesis.
SKELETAL DEVELOPMENT (E SCHIPANI AND E ZELZER, SECTION EDITORS)
Extracellular Matrix and Developing Growth Plate Johanna Myllyharju
Published online: 12 September 2014 # Springer Science+Business Media New York 2014
Abstract Growth plate is a specialized cartilaginous structure that mediates the longitudinal growth of skeletal bones. It consists of ordered zones of chondrocytes that secrete an extracellular matrix (ECM) composed of specific types of collagens and proteoglycans. Several heritable human skeletal dysplasias are caused by mutations in these ECM components and this review focuses on the roles of type II, IX, X, and XI collagens, aggrecan, matrilins, perlecan, and cartilage oligomeric matrix protein in the growth plate as deduced from human disease phenotypes and mouse models. Substantial advances have been achieved in deciphering the interaction networks and individual roles of these components in the construction of the growth plate ECM. Furthermore, ER stress and other cellular responses have been identified as key downstream effects of the ECM mutations contributing to abnormal growth plate development. The next challenge is to utilize the molecular level knowledge for the development of potential therapeutics. Keywords Growth plate . Extracellular matrix . Collagen . Aggrecan . Matrilin . Perlecan . Cartilage oligomeric matrix protein . ER stress
Introduction The majority of skeletal bones are formed via a process called endochondral ossification where an initial cartilaginous model is replaced by bone tissue [1–5]. Longitudinal growth of bones occurs via a specialized cartilaginous structure, the growth
J. Myllyharju (*) Oulu Center for Cell-Matrix Research, Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, PO Box 5000, FIN-90014 Oulu, Finland e-mail: [email protected]
plate. It consists of ordered zones of proliferating and differentiating chondrocytes (Fig. 1) that secrete a cartilaginous extracellular matrix (ECM) that is composed of specific types of collagens and proteoglycans. Ultimately, the growth plate chondrocytes differentiate into nondividing hypertrophic chondrocytes that undergo apoptosis and are replaced by bone cells (Fig. 1). The major collagen in growth plate cartilage is the fibril-forming type II collagen, other collagen types being the type XI and IX collagens. Type XI collagen also assembles into fibrils that are typically found within the core of the type II collagen fibrils, while type IX collagen, a fibril-associated collagen, resides at the surface of the type II/XI fibrils [6, 7]. Hypertrophic chondrocytes in the growth plate are characterized by the production of type X collagen. The growth plate cartilage also consists of a number of proteoglycans, such as aggrecan as the major component, perlecan, decorin, fibromodulin and lumican [1, 8, 9]. Other noncollagenous proteins of the growth plate cartilage include, eg, matrilins, COMP, tenascin-C, and link proteins [1, 8, 9]. The importance of the growth plate cartilage ECM is evident based on the existence of a number of human skeletal dysplasias caused by mutations in its components [10]. Besides the ECM components, several transcription factors, circulating hormones, growth factors, and cell surface receptors participate in the regulation of the growth plate cartilage [1–5, 11]. This review concentrates on the roles of the ECM components in growth plate cartilage as deduced from human disease phenotypes and mouse models. The effects of ECM component mutations can manifest by various mechanisms. The absence of a particular component can either be deleterious or tolerable, a mutant form of the component can lead to abnormal interactions and assembly of the ECM or the mutant form is not properly secreted from the endoplasmic reticulum (ER) and causes ER stress.
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ECM with extremely short and curved fibrils instead of a normal dense network of long type II collagen fibrils [28].
Type XI Collagen Type XI collagen is an α1(XI)α2(XI)α3(XI) heterotrimer. The α1(XI) and α2(XI) chains are encoded by the COL11A1 and COL11A2 genes, while the α3(XI) chain is encoded by the COL2A1 gene and is, thus, essentially identical to the α1(II) chain but has certain differences in its posttranslational modifications [13–16]. Collagen XI is found only in the thin collagen II fibrils and it is thought to regulate the fibril diameter [7, 12]. Mutations in the COL11A1 and COL11A2 genes have been identified in five distinct autosomal dominant or recessive skeletal disorders, examples being Stickler syndrome type 2 and Marshall syndrome with typical arthro-ophthalmologic features [10]. A spontaneous Col11a1 mutation that causes a premature stop codon exists in mice. Homozygous mice with this mutation have completely disorganized growth plates and severe chondrodysplasia and they die at birth [31]. Fig. 1 Schematic figure of the structure of growth plate
Type II Collagen Type IX Collagen The major collagen in growth plate cartilage is type II collagen that forms fibrils with thick (40 nm) and thin (16 nm) diameters [7, 12]. Type II collagen is a homotrimer composed of three identical α1(II) collagen chains encoded by the COL2A1 gene [13–16]. Mutations in the COL2A1 gene lead to at least ten autosomal dominant chondrodysplasias of varying severity, examples ranging from lethal forms (achondrogenesis type II and hypochondrogenesis) to diseases characterized by severe (spondyloepiphyseal dysplasia, Kniest dysplasia) or mild (Stickler syndrome) skeletal abnormalities [10]. The phenotype of type II collagen null mice resembles human achondrogenesis type II [17]. The Col2a1 null mice die immediately before or at birth, they are markedly smaller than their littermates, their long bones lack endochondral bone and the epiphyseal growth plate and intervertebral discs are not developed [17, 18]. Mice with various spontaneous or transgenic mutations in the Col2a1 gene typically display growth plate anomalies and chondrodysplasia [19–30]. For example transgenic mice with a dominant negative mutation in Col2a1 corresponding to a human mutation causing spondyloepiphyseal dysplasia have been shown to be neonatally lethal [28]. The mice had short bones, cleft palate, and respiratory failure. The proliferative chondrocytes of the growth plate were not arranged into distinct columns and thus, the proliferative zone was short and disorganized and accompanied with an enlargement of the prehypertrophic zone [28]. The epiphyseal cartilage of the mutant mice contained a loose
Type IX collagen is a heterotrimer consisting of three distinct collagen chains, α1(IX)α2(IX)α3(IX), encoded by the COL9A1, COL9A2, and COL9A3 genes [13–16]. Type IX collagen is associated to the surface of the type II/XI fibrils and is likely to regulate interactions between the fibrils themselves and with other ECM components. Mutations in the COL9A1, COL9A2, and COL9A3 genes are known to cause autosomal dominant multiple epiphyseal dysplasia and autosomal recessive Stickler syndrome [10]. Transgenic mice with a large in-frame deletion in Col9a1 suffer from mild chondrodysplasia and osteoarthritis [32]. Collagen IX null mice with inactivated Col9a1 gene were initially reported to have no obvious abnormalities at birth and apparently normal collagen fibrils were present in the rib cartilage [33, 34]. However, the mice developed osteoarthritis upon aging [33]. Later, it has been shown that lack of collagen IX has serious effects on the growth plate cartilage [35]. The columnar organization of chondrocytes was lost in the proliferative and hypertrophic zones of the mutant growth plate and its central regions were hypocellular [35]. It was also shown that glycosaminoglycan and β1 integrin distribution was abnormal and proliferation of chondrocytes was decreased in the mutant mice [35]. The mutant newborn mice also had a reduced length of the diaphysis of long bones, while the width was increased [35]. Interestingly, these abnormalities were attenuated in adult mice [35].
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Type X Collagen Type X collagen is an α1(X)3 homotrimer encoded by the COL10A1 gene [13–16]. It is specifically expressed in hypertrophic chondrocytes and assembles into hexagonal networklike structures [13–16]. Mutations in COL10A1 lead to autosomal dominant Schmid metaphyseal dysplasia [10]. Transgenic mice expressing a dominant negative mutant form of chicken collagen X have skeletal and hematopoietic defects [36, 37]. Endochondral ossification was affected in the mutant mice and was manifested as variable skeletal phenotypes ranging from thoracolumbar kyphosis and lethality at about 3 weeks of age to variable degrees of dwarfism with susceptibility to skeletal deformities. The hypertrophic zone of the growth plate was compressed in all the mutant mice, the degree of compression correlating with the severity of the phenotype [36, 37]. In contrast, analysis of the first collagen X null mouse line generated showed no obvious abnormalities in the development and growth of long bones [38]. However, phenotypic changes partly resembling Schmid metaphyseal dysplasia were observed in a second independently generated collagen X null mouse line [39]. The height of the resting zone of growth plate chondrocytes was reduced and the architecture of trabecular bone was altered in the chondro-osseous junction in the collagen X null mice [39]. In addition, differences in the distribution and organization of growth plate ECM components were observed [39]. Interestingly, it was later noticed that about 11 % of the collagen X null mice from the line generated first do not survive beyond 3 weeks of age [40]. In the perinatal lethal collagen X null mice, compression of the proliferative zone of the growth plate was observed and trabecular bone was reduced [40]. Transgenic mice expressing collagen X containing a deletion equivalent to a mutation found in human Schmid metaphyseal dysplasia patients suffer from dwarfism and have short limbs [41, 42]. The mutant collagen X was found to accumulate within the hypertrophic chondrocytes in which ER stress was observed [42]. However, the hypertrophic chondrocytes were not directed to an apoptotic pathway but utilized adaptive mechanisms including changes in cell cycle regulation, reversion to a prehypertrophic state and altered terminal differentiation [42]. Knock-in mice with a collagen X Asn617Lys mutation corresponding to a human mutation found in the Schmid metaphyseal dysplasia also had short limbs [43]. The hypertrophic zone of the growth plate was expanded, and the hypertrophic chondrocytes retaining the mutant collagen X displayed ER stress and unfolded protein response [43].
Aggrecan Aggrecan is a large chondroitin sulfate proteoglycan and is the major proteoglycan component of cartilage [5, 9]. The
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chondroitin sulfate side chains of aggrecan generate a high negative charge density and create an osmotic environment that retains water, which provides cartilage tissue with high compressive endurance. Human mutations in aggrecan cause two types of spondyloepiphyseal dysplasia, the autosomal dominant Kimberley type and autosomal recessive Aggrecan type, and autosomal dominant familial osteochondritis dissecans [10]. A natural short deletion in the mouse gene encoding aggrecan that leads to a truncated protein causes an autosomal recessive cartilage matrix deficiency syndrome with abnormal craniofacial structures, short limbs and tail and perinatal lethality [44]. The organization of chondrocytes is severely disrupted, the amount of hypertrophic chondrocytes is significantly reduced and expression of other ECM genes is altered in the growth plates of the mutant mice [45].
Matrilins Matrilins are multi-domain proteins consisting of von Willebrand factor A domain(s), epidermal growth factor-like motif(s) and a C-terminal coiled-coil domain that is responsible for the homo- and hetero-oligomerization of the matrilins [46]. The matrilin family consists of matrilins 1–4, of which matrilins 1 and 3 are mainly found in cartilage, matrilins 2 and 4 being detected also in other tissues [46]. Matrilins associate with cartilage collagen fibrils and bind covalently to aggrecan and are, thus, regarded as adaptor proteins in ECM assembly [46]. Mutations in the human gene encoding matrilin-3 have been identified in autosomal dominant multiple epiphyseal dysplasia type 5 and in autosomal recessive spondyloepi metaphyseal dysplasia Matrilin type [10]. No obvious abnormalities in skeletal development and cartilage ECM were initially discovered in matrilin-1, -2, and -3 knockout mice suggesting mutual compensatory capacity [47–49]. However, despite normal skeletogenesis and growth plate structure, abnormal fibrillogenesis of type II collagen and fibril organization at the maturation zone of the growth plate was observed in matrilin-1 knockout mice [50] and premature maturation of chondrocytes to prehypertrophic and hypertrophic chondrocytes and expansion of the hypertrophic zone of the growth plate was observed in matrilin-3 knockout mice with increased bone mineral density and susceptibility to osteoarthritis [51]. Furthermore, matrilin-1/matrilin-3 double null mice have no apparent skeletal abnormalities, although certain changes were observed in the cartilage ECM including increased collagen fibril diameters and collagen volume density in the epiphysis and growth plate [52]. Interestingly, knock-in mice with a matrilin-3 mutation (Val194Asp) known to cause multiple epiphyseal dysplasia in humans, suffer from progressive dysplasia and short-limbed dwarfism [53]. The mutant matrilin-3 was found to be retained within the ER and
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associated with induction of the unfolded protein response [53, 54]. Furthermore, proliferation of the growth plate chondrocytes was reduced and apoptosis was spatially dysregulated [53]. Combination with a null matrilin-1 mutation did not enhance the skeletal abnormalities observed in the Val194Asp matrilin-3 mutant mice [55].
Perlecan Perlecan is a large heparan sulfate/chondroitin sulfate proteoglycan and is a ubiquitous component of basement membranes, articular cartilage and growth plate [56–58]. The large protein core of perlecan consists of five domains that contain a sperm-agrin-enterokinase module and repeats similar to those present in the low-density lipoprotein receptor, immunoglobulin, laminin and epidermal growth factor, ie, molecules involved in eg, lipid metabolism, cell proliferation, and adhesion [56, 57]. Perlecan interacts with a large variety of molecules including growth factors and their receptors, integrins, and various ECM components and it has been shown to be involved in eg, angiogenesis, cardiovascular development and neurogenesis [56–58]. In addition, perlecan is essential for cartilage and bone formation. Mutations in the human perlecan gene cause three autosomal recessive skeletal disorders, the Silverman-Handmaker type and RollandDesbuquois type dyssegmental dysplasias and the SchwartzJampel syndrome (myotonic chondrodystrophy) [10]. Many of the perlecan null mice are embryonic lethal around midgestation because of abnormal cephalic development or heart failure [59, 60]. The surviving embryos have progressive defects in skeletal development starting at E14.5 when endochondral ossification begins and the mice die shortly after birth [59, 60]. The zonal and columnar arrangement of chondrocytes was extremely disorganized in the growth plate and endochondral ossification was reduced, type II collagen fibrils were sparse and disorganized and the proliferation of chondrocytes was reduced [59, 60]. It has been shown that the chondroitin sulfate side chains of perlecan enhance fibril formation of type II collagen fibrils [61]. Furthermore, the absence of perlecan was also found to affect fibroblast growth factor signaling in the growth plate [59]. Knock-in perlecan mutant mice with a Cys1532Tyr mutation have been generated to model the Schwartz-Jampel syndrome [62]. However, the knock-in mice had no obvious anatomic phenotype and the mutation did not affect the transcription level, secretion, or deposition of perlecan [62]. Interestingly, when the neomycin selection cassette was retained in the Cys1532Tyr mutant mice, they displayed skeletal abnormalities closely resembling the disease phenotype [62, 63]. In these mice, transcription of the mutant perlecan gene was altered (reduced level of fulllength mutant perlecan mRNA and emergence of a truncated transcript) and secretion of perlecan was reduced [62].
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Perlecan has also been shown to regulate VEGF signaling and to be essential for vascularization during endochondral bone formation [64].
Cartilage Oligomeric Matrix Protein Cartilage oligomeric matrix protein (COMP, thrombospondin 5) is a pentameric protein composed of five identical subunits that are linked via their N-terminal coiled-coil domain, which is followed by EGF-like and thrombospondin type 3 repeats and a C-terminal globular domain that can interact with several other ECM proteins including type I, II, and IX collagens, matrilins and fibronectin [4, 9, 65]. Mutations in the human gene coding for COMP cause autosomal dominant pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia type 1 [10]. The COMP mutations act in a dominant negative manner and cause retention of the mutant COMP polypeptides in the ER leading to unfolded protein response, cell death, and reduction of chondrocytes in the growth plate [65]. In addition to intracellular retention of COMP, studies with PSACH mutant chondrocytes showed that the COMP mutations simultaneously reduced secretion of type IX collagen and matrilin-3, but not that of type II collagen or aggrecan, leading to marked reduction in collagen IX and matrilin-3 in the matrix [66]. As in the case of matrilin-3 null mice, COMP null mice seem to have normal skeletal development [67] and simultaneous deficiency of COMP did not aggravate the phenotype of collagen IX null mice [68]. In contrast, mice harboring PSACH mutations in COMP manifest several of the disease characteristics [65]. Transgenic mice expressing a COMP mutant with a deletion of Asp469 showed slight growth retardation in the males, abnormal columnar organization of chondrocytes in the growth plate with atypical collagen fibrils and loss of proteoglycans and ER stress [69]. Although PSACH is an autosomal dominant disorder, heterozygous mice with a knock-in Asp469del COMP mutation showed no obvious skeletal defects, but the homozygous Asp469del mutant mice had progressive short-limb dwarfism and disorganized chondrocyte columns in the growth plate with hypocellular areas in the resting and upper proliferative zones [70]. The mutant COMP was retained within the ER together with matrilin-3 and to some extent with collagen IX with concomitant reduction in their extracellular deposition and ultrastructural abnormalities of the ECM [70]. Interestingly, although dilation of the ER was detected, unfolded protein response was not induced but instead changes in the expression of genes involved in oxidative stress, apoptosis, and cell cycle arrest were observed [70]. Heterozygous knockin mice with a COMP Thr583Met mutation causing mild PSACH or multiple epiphyseal dysplasia type 1 also had no significant changes in their skeletogenesis, but the homozygous mice developed short-limb dwarfism by 9 weeks of age
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[71]. The chondrocytes in the growth plate were sparse, their columnar organization was disturbed from 2 weeks of age and their proliferation was decreased and apoptosis was increased [71]. Unfolded protein response was detected in the chondrocytes, but significant amounts of the mutant COMP were still secreted into the growth plate ECM, which showed ultrastructural abnormalities and changes in the localization of the interacting proteins matrilin-3 and collagen IX [71]. In all these mouse lines with disease-causing COMP mutations the phenotype has been milder than in the corresponding human patients. A mouse line that more closely models the human PSACH phenotype at the cellular and growth plate level has been generated using a tetracycline-inducible transgene approach and used to analyze the disease causing mechanisms in detail at cellular level [72–74].
ECM Mutations and ER Stress As indicated above ER stress with or without induction of the unfolded protein response is commonly observed in growth plate chondrocytes expressing mutant ECM components and is typically associated with changes in chondrocyte proliferation and apoptosis [75]. Similarly, it has been shown that chondrocytes with reduced amount of collagen prolyl 4-hydroxylase, an essential enzyme of collagen synthesis [13], leads to accumulation of ECM components within the cells and ER stress [76]. To study the contribution of chondrocyte ER stress to abnormal skeletogenesis independent of any ECM mutations, a transgenic mouse line expressing mutant thyroglobulin specifically in the chondrocytes has been generated [77••]. The mutant thyroglobulin is known to cause ER stress in thyrocytes and was found to cause short-limb dwarfism in the transgenic mice [77••]. The ECM of the growth plates of the transgenic mice was apparently normal, but the chondrocyte columns were disordered with hypocellular areas [77••]. Chondrocyte proliferation was reduced and showed moderate cellular stress without induction of the unfolded protein response or apoptosis [77••]. These data show that ER stress is sufficient to cause abnormal skeletogenesis. ER stress can be alleviated chemically by 4-sodium phenylbutyrate and its effect has been studied on mice expressing the Val194Asp mutant matrilin-3 or the Asn617Lys mutant collagen X [54]. However, no increases in the secretion of the mutant proteins or improvement of the disease phenotype were observed, the lack of efficacy being potentially because of the avascularity of the growth plate cartilage [54].
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critical roles in the survival, differentiation, and function of growth plate chondrocytes and thus, normal skeletogenesis. Significant progress has been made in understanding the interaction networks of the ECM components and their individual roles in the assembly of the growth plate ECM. Furthermore, ER stress and other cellular responses have been identified as key downstream effects of the ECM mutations contributing to abnormal growth plate development. The challenge in the future is to utilize the current molecular level knowledge for the development of potential therapeutics. Compliance with Ethics Guidelines Conflict of Interest J. Myllyharju has received research grants from FibroGen Inc. Human and Animal Rights and Informed Consent All studies by the authors involving animal and/or human subjects were performed after approval by the appropriate institutional review boards. When required, written informed consent was obtained from all participants.
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Conclusions Based on evidence from human heritable diseases and genemodified animal models, certain ECM components have
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