Biochemical, Genetic, and Molecular Interactions in Development - part 3 pdf

72 Takeda et al. Generation of Transgenic Mice Plasmids were digested with appropriate restriction enzymes and inserts were purified by agarose gel electrophoresis. Linear DNA fragments were microinjected into pronuclei of fertilized C57BL/ 6SnJ mouse oocytes that were subsequently reimplanted into oviducts of pseudopregnant CD1 foster mothers (Jackson Laboratories). _1(II) Cbfa1, _1(II) Cbfa1a, and _I(II) Cbfa16PST transgenes were respectively coinjected with the 1.3kb of OG2-LacZ construct to obtain transgenic mice coexpressing the two transgenes. Genotypes were determined by polymerase chain reaction (PCR) using the fol- lowing as primers: 5'-GGCAGCACGCTATTAAATCCAA-3' and 5'-GGTTTCAGGGGGAGGTGTG GGAGG-3' for the _1(II) Cbfa1 mice; 5'-CTGGACATCATAGCAAAGGCCC-3' and 5'-GGTTTCAG GGGGAGGTGTGGGAGG-3' for the _1(II) Cbfa1a mice; and 5'-CGGAGCGGACGAGGCAAGA GTTTC-3' and 5'-GGTTTCAGGGGGAGGTGTGGGAGG-3' for the _I(II) Cbfa16PST mice. Sex was determined by PCR using the Sry–specific primers 5'-CATGACCACCACCACCACCAA-3' and 5'-TC ATGAGACTGCCAACCACAG-3' (25). Reverse Transcription PCR Analysis To monitor the transgene expression, total RNA was prepared from 12.5-dpc embryos. Three to four embryos were analyzed independently for each genotypes. RNA extraction, cDNA synthesis, and PCR amplification were performed using standard protocols (26). Exon 2 amplification of the HPRT gene was used as internal control of the quantity and quality of the cDNAs. The following sets of the primers were used: transgene specific PCR, 5'-CCAGGCAGTTCCCAAGCATT-3' and 5'-AGAG CTATGACGTCGCATGCACAC-3'; endogenous Cbfa1, 5'-GGCAGCACGCTATTAAATCCAAA-3' and 5'-TGACTGCCCCCACCCTCTTAG-3'; and Hprt, 5'-GTTGAGAGATCATCTCCACC-3' and 5'-AGC GATGATGAACCAGGTTA-3'. Fig. 11. Schematic representation of the roles of Cbfa1 in endochondral ossification. Cbfa1 favors chondro- cyte hypertrophy via an Ihh-dependent pathway. In turn, Ihh induces differentiation of the cells of the bone collar through a Cbfa1-dependent pathway. Cbfa1 also favors VEGF expression. Cbfa1 Controls Chondrocyte Hypertrophy 73 Skeletal Preparation Mice were dissected, fixed in 100% ethanol overnight, then stained in alcian blue dye solution (0.015% alcian blue 8GX [Sigma], 20% acetic acid, 80% ethanol) overnight and transferred to 2% potassium hydroxide for 24 h or longer, dependent on the age of the mice. Subsequently, they were stained in alizarin red solution (0.005% Alizarin sodium sulfate [Sigma], 1% KOH) and cleared in 1% KOH/20% glycerol. Histological Analyses and In Situ Hybridization Tissues were fixed in 4% paraformaldehyde/phosphate-buffered saline overnight at 4°C and decal- cified in 25% EDTA at 37°C for 3 d when older than newborn. Specimens were embedded in paraffin and sectioned at 6 µm. For histological analysis, sections were stained with alcian blue (1% alcian blue 8GX, 3% acetic acid) and counterstained with eosin. For alkaline phosphatase/TRAP staining, sections were first stained for alkaline phosphatase with Fast blue BB (Sigma) then for TRAP with pararosanil- ine (Sigma) following established conditions (27). Gelatinase assay was performed as described (28). In situ hybridization was performed using complementary 35 S-labeled riboprobes. Cbfa1 and _ I(II) collagen probes have been previously described (15). The Ihh probe is a 540-bp fragment of Ihh 3' un- translated region. The _ I(X) collagen probe was obtained from Dr. B.R. Olsen (Harvard Medical School, Boston, MA). Hybridizations were performed overnight at 55°C, and washes were performed at 63°C. Autoradiography and Hoechst 33528 staining were performed as described (29). LacZ Staining and Immunohistochemistry Skinned and eviscerated animals were fixed in 1% paraformaldehyde, 0.2% glutalaldehyde in phos- phate buffer (pH 7.3) for 45 min, and stained overnight with X-Gal (5-bromo-4-chloro-3indoyl `-D- galactosidase). Specimens were embedded in paraffin and sectioned at 6 µm. Sections were counter- stained with eosin. Immunohistochemistry was performed according standard protocol (26). Anti-VEGF antibody was purchased from Santa Cruz Biotechnology. BrdU Labeling Mice were injected intraperitoneally with 10 <4 mM BrdU/g body weight 1 h before sacrifice. Tibiae were dissected, fixed, decalcified, and embedded in paraffin as previously. BrdU was detected using a Zymed kit following the manufacturer’s protocol (Zymed). BrdU-positive cells present in the growth plate of at least five different sections were counted for both wt and _I(II) Cbfa1 mice. Statistical dif- ferences between groups were assessed by Student’s t-test. DNA Transfection Assays F9 cells were transfected with 5 µg of empty or Cbfa1 or Cbfa1a expression vector (15), 5 µg of p6OSE2-luc reporter vector (23), and 2 µg of pSV`gal plasmid. Transfections, luciferase assays, and `-galactosidase assays were performed as described (23). Data represent ratios of luciferase/`-galac- tosidase activities and values are means of six independent transfection experiments. ACKNOWLEDGMENTS The authors are indebted to J. Liu and J. Shen for their superb technical assistance and their com- mitment to this study. The authors also thank Dr. Chung, Kronenberg, McMahon, and Olsen for in situ hybridization probes. They are grateful to Dr. G. Friedrich and members of the Karsenty labora- tory for critical reading of the manuscript. This work was supported by March of Dimes FY99-489 and NIH R01 AR45548, NIH P01 AR42919 and Eli Lilly grants to G.K.; Arthritis Foundation and March of Dimes FY99-761 grants to P.D.; and Arthritis Foundation Postdoctoral Fellowship to S.T. 74 Takeda et al. REFERENCES 1. Horton, W. A. (1993) Morphology of connective tissue: Cartilage, in Connective tissue heritable disorders, Wiley- Liss, Inc., New York, pp. 73–84. 2. Caplan, A. I. and Pechak, D. G. (1987) The cellular and molecular embryology of bone formation, in Bone and mineral research. Vol. 5. (Peck, W. A., ed.), Elsevier, New York, pp. 117–183. 3. Linsenmayer, T. F., Chen, Q. A., Gibney, E., Gordon, M. K., Marchant, J. K., Mayne, R., et al. (1991) Collagen type IX and X in the developing chick tibiotarsus: analyses of mRNAs and proteins. Development 111, 191–196. 4. Mundlos, S. (1994) Expression patterns of matrix genes during human skeletal development. Prog. Histochem. Cyto- chem. 28, No. 3. 5. Poole, A. R. 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Cytol. 159, 265–358. 38. Chung, U. I., Lanske, B., Lee, K., Li, E., and Kronenberg, H. (1998) The parathyroid hormone/parathyroid hormone- related peptide receptor coordinates endochondral bone development by directly controlling chondrocyte differentia- tion. Proc. Natl. Acad. Sci. USA 95, 13030–13035. 76 Takeda et al. Molecular Biology of Collagens 77 77 From: The Skeleton: Biochemical, Genetic, and Molecular Interactions in Development and Homeostasis Edited by: E. J. Massaro and J. M. Rogers © Humana Press Inc., Totowa, NJ 5 Molecular Biology and Biosynthesis of Collagens Johanna Myllyharju INTRODUCTION The collagens are a heterogeneous family of extracellular matrix proteins that have a major role in maintaining the structural integrity of various tissues and organs, although they also have many other important biological functions. Collagens are the most abundant proteins in the human body, with approx 30% of protein mass consisting of collagen. Tissues that are especially rich in collagens are bone, skin, tendon, cartilage, ligaments, and vascular walls. The extracellular matrix in bone and tendon consists of up to 90% of collagen and that of skin approx 50%. The collagen superfamily now includes at least 27 collagen types and more than 15 additional proteins that have collagen-like domains. Most collagens form polymeric assemblies, and the superfamily can be divided into several classes based on their supramolecular structures or other features. Biosynthesis of collagens is a complex process that requires eight specific post-translational enzymes. Collagens have an important role in the healing of wounds and fractures and, thus, inhibition of collagen synthesis will delay healing. However, exces- sive collagen formation can lead to fibrosis, thus impairing the normal functioning of the affected organ. The essential function of collagens is illustrated by the wide variety of disease phenotypes caused by mutations in their genes. THE COLLAGEN SUPERFAMILY At least 27 proteins with altogether 42 distinct polypeptide chains and corresponding genes are now known as collagens (refs. 1–8; Table 1). Collagens are extracellular matrix proteins that consist of three polypeptide chains, called _ chains, and contain at least one unique triple-helical domain with repeating -Gly-X-Y- sequences in each of the constituent chains. The presence of glycine, the small- est amino acid, in every third position in the triple-helical domain is critical because a larger amino acid does not fit into the restricted space in the centre of the triple helix. The X- and Y-position amino acids vary according to the collagen type and domain, but proline is frequently found in the X posi- tion and 4-hydroxyproline in the Y position. 4-Hydroxyproline residues have an important role in the thermal stability of the triple helix (9). Depending on the collagen type, the _ chains differ in length and in the number of possible interruptions in the triple helix (Fig. 1). In some collagen types, all the three _ chains are identical, whereas in others the collagen molecule consists of two or three different _ chains (Table 1). The collagen superfamily can be classified into eight groups based on their poly- meric structures or other features (Fig. 1): A, fibril-forming collagens, types I–III, V, XI, XXIV, and XXVII; B, fibril-associated collagens with interrupted triple-helices (FACIT collagens), types IX, XII, XIV, XVI, XIX–XXII, and XXVI; C, collagens forming hexagonal networks, types VIII and X; D, the 78 Myllyharju Table 1 Collagen Types, Their Constituent Polypeptide Chains, Genes, and Occurrence in Tissues a Type Constituent Gene Occurrence I _1(I) COL1A1 Most connective tissues, especially in dermis, bone, tendon, ligament _2(I) COL1A2 II _1(II) COL2A1 Cartilage, intervertebrate disc, inner ear, vitreous humour, cornea III _1(III) COL3A1 As type I collagen except absent in bone and tendon. Abundantly expressed in elastic tissues, such as skin, inner organs, and blood vessels IV _1(IV) COL4A1 All basement membranes _2(IV) COL4A2 _3(IV) COL4A3 _4(IV) COL4A4 _5(IV) COL4A5 _6(IV) COL4A6 V _1(V) COL5A1 Tissues containing type I collagen _2(V) COL5A2 _3(V) COL5A3 _4(V) COL5A4 Nervous system VI _1(VI) COL6A1 Most connective tissues _2(VI) COL6A2 _3(VI) COL6A3 VII _1(VII) COL7A1 Anchoring fibrils in skin, cornea, cervix, oral, and esophageal mucosa VIII _1(VIII) COL8A1 Many tissues _2(VIII) COL8A2 IX _1(IX) COL9A1 Tissues containing type II collagen _2(IX) COL9A2 _3(IX) COL9A3 X _1(X) COL10A1 Hypertrophic cartilage XI _1(XI) COL11A1 Tissues containing type II collagen _2(XI) COL11A2 _3(XI) b COL2A1 XII _1(XII) COL12A1 Tissues containing type I collagen XIII _1(XIII) COL13A1 Many tissues XIV _1(XIV) COL14A1 Tissues containing type I collagen XV _1(XV) COL15A1 Many tissues in the basement membrane zone XVI _1(XVI) COL16A1 Many tissues XVII _1(XVII) COL17A1 Skin hemidesmosomes XVIII _1(XVIII) COL18A1 Many tissues in the basement membrane zone XIX _1(XIX) COL19A1 Many tissues in the basement membrane zone XX _1(XX) COL20A1 Many tissues XXI _1(XXI) COL21A1 Many tissues XXII _1(XXII) c COL22A1 XXIII _1(XXIII) COL23A1 Metastatic tumor cells XXIV _1(XXIV) COL24A1 Developing bone and cornea XXV _1(XXV) COL25A1 Neurons XXVI _1(XXVI) COL26A1 Testis, ovary XXVII _1(XXVII) COL27A1 Cartilage, eye, ear, and lung a See refs. 1–8. b The _3(XI) is a post-translational variant of _1(II). c Complete cDNA sequence characterized (M. Koch, M. Gordon, and R. E. Burgeson, personal communication). Molecular Biology of Collagens 79 Fig. 1. Schematic representation of various members of the collagen superfamily and their known supramolecular assemblies. The letters refer to the families described in the text. The supramolecular assemblies of families G and H have not been elucidated and are hence not sh own. The closed circles indicate N- and C-terminal noncollagenous domains, whereas open circles indicate noncollagenous domains interrupting the collagen triple helix. GAG, glycosami- noglycan; PM, plasma membrane. Modified from ref. 1 with permission. 79 80 Myllyharju family of type IV collagens found in basement membranes; E, type VI collagen that forms beaded filaments; F, type VII collagen that forms anchoring fibrils for basement membranes; G, collagens with transmembrane domains, types XIII, XVII, XXIII, and XXV; and H, the family of type XV and XVIII collagens (1–3). The most abundant type I–III collagens, in addition to type V and XI collagens, self-assemble into long quarter-staggered fibrils and are thus called fibril-forming collagens (Fig. 1; ref. 1). The fibril- forming collagens contain large triple-helical domains of about 1000 amino acids with continuous -Gly-X-Y- repeats and short nontriple-helical N and C telopeptides at both ends. The telopeptides are the primary sites for intermolecular crosslinking, which is important for the stabilization of the collagen fibrils (10). These collagens are first synthesized as larger precursors, procollagens, that Fig. 2. Biosynthesis of a fibril-forming collagen. Procollagen polypeptide chains are synthesized on the ribo- somes of the rough endoplasmic reticulum and secreted into the lumen, where the chains are modified by hydroxy- lation of certain proline and lysine residues and glycosylation before chain association and triple helix formation. The newly formed procollagen molecules are secreted into the extracellular space, where the N and C propeptides are cleaved by specific proteinases. The collagen molecules thus generated spontaneously assemble into fibrils, which are stabilized by the formation of covalent crosslinks. Reproduced from ref. 1 with permission. Molecular Biology of Collagens 81 have globular N and C propeptide domains, which are cleaved off from the mature collagen molecules (Figs. 1 and 2; ref. 1). Type I collagen is the major structural constituent of most connective tissues, including bone, whereas type II is the major component in cartilage (Table 1). Type III collagen is generally found in the same tissues as type I, but especially in elastic tissues (Table 1). Collagen fibrils are often hetero- geneous, containing more than one collagen type. Type I collagen fibrils usually contain small amounts of type III, V, and XII, with type V being located in the core and types III and XII on the surface of the fibril (1). The cartilage collagen fibrils have type II as their main component, with a core of type XI and a surface of type IX (1). The type V and XI collagens have an important role in the regulation of the type I and type II fibril diameters, respectively (11,12). BIOSYNTHESIS OF COLLAGENS Biosynthesis of collagens is a complex process that involves a number of intracellular and extra- cellular post-translational modifications (1,13,14). The fibril-forming collagens are synthesized as larger precursors that have globular propeptide domains at both their N and C-terminal ends (Fig. 2). An N- terminal signal sequence targets the nascent pro_ chains into the endoplasmic reticulum (ER), where a series of modifications occur. The main intracellular modifications (Fig. 2) of the pro_ chains include the cleavage of the signal peptide; hydroxylation of specific proline and lysine residues to 4-hydroxy- proline, 3-hydroxyproline, and hydroxylysine; O-linked glycosylation of some of the hydroxylysine residues to galactosylhydroxylysine and glucosyl galactosylhydroxylysine; N-linked glycosylation of one or both of the propeptides; and formation of intrachain and interchain disulfide bonds (1,13,14). After the C propeptides have associated in a type-specific manner (13) and approx 100 proline residues in each chain have been hydroxylated, a nucleation site for triple helix formation is formed in the C- terminal end of the triple-helical domain and the triple helix is then propagated toward the N terminus. The procollagen molecules are transported from the ER through the Golgi complex by progres- sive maturation of the Golgi cisternae rather than vesicular transport (15). The extracellular steps (1) involve the conversion of procollagen molecules to collagen molecules by the cleavage of the N and C propeptides (16), self-assembly of the collagen molecules into fibrils by nucleation and propaga- tion, and formation of covalent crosslinks (10). The collagen synthesis described above is characteristic for fibril-forming collagens. The biosynthe- sis steps of nonfibrillar collagens are principally the same with certain exceptions (1). Many collagens have globular N- and/or C-terminal domains that are not cleaved (Fig. 1), the triple helices of transmem- brane collagens are probably propagated from the N to the C terminus (17,18), and the triple helices of some collagens are modified by N-linked glycosylation or addition of glycosaminoglycan side chains. The intracellular modifications require five specific enzymes: three collagen hydroxylases (19– 21) and two collagen glycosyltransferases (1), whereas the extracellular modifications require three specific enzymes: two proteinases that cleave the propeptides (16) and an oxidase (22) that converts certain lysine and hydroxylysine residues to reactive aldehyde derivatives required in the crosslink formation. The collagen hydroxylases, prolyl 4-hydroxylase, prolyl 3-hydroxylase, and lysyl hydrox- ylase, catalyze the formation of 4-hydroxyproline, 3-hydroxyproline, and hydroxylysine residues in -X-Pro-Gly, -Pro-4Hyp-Gly-, and -X-Lys-Gly- triplets, respectively (19–21). 4-Hydroxyproline resi- dues have an important role in stabilizing the collagen triple helix (9) and hydroxylysine residues serve as attachments sites for carbohydrate units and participate in the formation of intermolecular collagen crosslinks (19). The function of 3-hydroxyproline residues is still unknown (19). The specific collagen-modifying enzymes were long assumed to be of one type only, with no iso- enzymes, but this concept has changed recently. Vertebrate prolyl 4-hydroxylases are now known to have at least three isoenzymes (19–21,23,24). Type I prolyl 4-hydroxylase is the main form in most cell types, whereas the type II enzyme is the major form in chondrocytes, osteoblasts, endothelial [...]... C428–C 432 39 Shyy, J Y and Chien, S (1997) Role of integrins in cellular responses to mechanical stress and adhesion Curr Opin Cell Biol 9, 707–7 13 40 MacKenna, D A., Dolfi, F., Vuori, K., and Ruoslahti, E (1998) Extracellular signal-regulated kinase and c-Jun NH2terminal kinase activation by mechanical stretch is integrin-dependent and matrix-specific in rat cardiac fibroblasts J Clin Invest 101, 30 1 31 0... The Skeleton: Biochemical, Genetic, and Molecular Interactions in Development and Homeostasis Edited by: E J Massaro and J M Rogers © Humana Press Inc., Totowa, NJ 89 90 Chen Fig 1 Diagram depicting chondrocyte differentiation process Type II, type II collagen; AGG, aggrecan; LP, link protein; Mat -3 , matrilin -3 ; Mat-1, matrilin-1; CMP, cartilage matrix protein; Type X, type X collagen The increase of... 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Heinegard, D., and Morgelin, M (1996) Interaction of cartilage matrix protein with aggrecanincreased covalent cross-linking with tissue maturation J Biol Chem 271, 32 247 32 252 33 Paulsson, M and Heinegard, D (1979) Matrix proteins bound to associatively prepared proteoglycans from bovine cartilage Biochem J 1 83, 539 –545 34 Ando, J., Ohtsuka, A., Korenaga, R., Kawamura, T., and Kamiya, A (19 93) Wall shear... The other two MAP kinase pathways, Jun-N-terminal kinase and p38, are not activated primarily by mitogens but by cellular stress and inflammatory cytokines (42, 43) We have obtained direct evidence that ERK and p38 are greatly activated by matrix deformation in our 3D culture system, whereas Jun-Nterminal kinase is only minimally activated The activation of ERK and p38 accompanies the stimulation of chondrocyte... 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Micron 32 , 2 23 237 12 Blaschke, U K., Eikenberry, E F., Hulmes, D J., Galla, H J., and Bruckner, P (2000) Collagen XI nucleates selfassembly and limits lateral growth of cartilage fibrils J Biol Chem 275, 1 037 0–1 037 8 13 McLaughlin, S H and Bulleid, N J (1998) Molecular recognition in procollagen chain assembly Matrix Biol 16, 36 9 37 7 14 Lamandé, S R and Bateman, J F (1999) Procollagen folding and assembly:... dysfunction and perinatal death in mice Circulation 106, 25 03 2509 37 Heikkinen, J., Risteli, M., Wang, C., Latvala, J., Rossi, M., Valtavaara, M., and Myllylä, R (2000) Lysyl hydroxylase 3 is a multifunctional protein possessing collagen glucosyltransferase activity J Biol Chem 275, 36 158 36 1 63 38 Wang, C., Risteli, M., Heikkinen, J., Hussa, A.-K., Uitto, L., and Myllylä, R (2002) Identification of amino... stretch In contrast, a calcium channel blocker nifedipine inhibited both the stretchinduced proliferation and the increase of matrilin-1 mRNA This suggests that stretch-induced matrix deformation regulated chondrocyte proliferation and differentiation via two signal transduction pathways, with stretch-activated channels involved in transducing the proliferative signals, and calcium channels involved in . prolyl 4-hydroxylase, prolyl 3- hydroxylase, and lysyl hydrox- ylase, catalyze the formation of 4-hydroxyproline, 3- hydroxyproline, and hydroxylysine residues in -X-Pro-Gly, -Pro-4Hyp-Gly-, and -X-Lys-Gly-. chains, called _ chains, and contain at least one unique triple-helical domain with repeating -Gly-X-Y- sequences in each of the constituent chains. The presence of glycine, the small- est amino. eviscerated animals were fixed in 1% paraformaldehyde, 0.2% glutalaldehyde in phos- phate buffer (pH 7 .3) for 45 min, and stained overnight with X-Gal (5-bromo-4-chloro-3indoyl `-D- galactosidase). Specimens