Matrix proteins associated with bone calcification are present in human vascular smooth muscle cells grownin vitro

Summary Atherosclerotic lesions are composed of cellular elements that have migrated from the vessel lumen and wall to form the cellular component of the developing plaque. The cellular elements are influenced by various growth-regulatory molecules, cytokines, chemoattractants, and vasoregulatory molecules that regulate the synthesis of the extracellular matrix composing the plaque. Because vascular smooth muscle cells (VSMC) constitute the major cellular elements of the atherosclerotic plaque and are thought to be responsible for the extracellular matrix that becomes calcified in mature plaques, immunostaining for collagenous and noncollagenous proteins typically associated with bone matrix was conducted on VSMC grownin vitro. VSMC obtained from human aorta were grown in chambers on glass slides and immunostained for procollagen type I, bone sialoprotein, osteonectin, osteocalcin, osteopontin, decorin, and biglycan. VSMC demonstrated an intense staining for procollagen type I, and a moderately intense staining for the noncollagenous proteins, bone sialoprotein and osteonectin, two proteins closely associated with bone mineralization. Minimal immunostaining was noted for osteocalcin, osteopontin, decorin, and biglycan. The presence in VSMC of collagenous and noncollagenous proteins associated with bone mineralization suggest that the smooth muscle cells in the developing atherosclerotic plaque play an important role in the deposition of the extracellular matrix involved in calcification of developing lesions.     

Effects of streptozotocin-induced diabetes and physical training on gene expression of extracellular matrix proteins in mouse skeletal muscle

Diabetes induces changes in the structure and function of the extracellular matrix (ECM) in many tissues. We investigated the effects of diabetes, physical training, and their combination on the gene expression of ECM proteins in skeletal muscle. Mice were divided to control (C), training (T), streptozotocin-induced diabetic (D), and diabetic training (DT) groups. Training groups (T, DT) performed 1, 3, or 5 wk of endurance training on a treadmill. Gene expression of calf muscles was analyzed using microarray and quantitative PCR. Training group samples were collected 24 h after the last training session. Diabetes affected the gene expression of several collagens (types I, III, IV, V, VI, and XV), some noncollagenous glycoproteins, and proteoglycans (e.g., elastin, thrombospondin-1, laminin-2, decorin). Reduced gene expression of collagens in diabetic skeletal muscle was partially attenuated as a result of physical training. In diabetes, mRNA expression of the basement membrane (BM) collagens decreased and that of noncollagenous glycoproteins increased. This may change the structure of the BM in a less collagenous direction and affect its properties.     

Effects of cis-4-hydroxy-L-proline, and inhibitor of Schwann cell differentiation, on the secretion of collagenous and noncollagenous proteins by Schwann cells

The proline analog cis-4-hydroxy-L-proline (CHP) was previously shown to inhibit both Schwann cell (SC) differentiation and extracellular matrix (ECM) formation in cultures of rat SCs and dorsal root ganglion neurons. We confirmed that CHP inhibits basal lamina formation by immunofluorescence with antibodies to laminin, type IV collagen, and heparan sulfate proteoglycan. In order to test the hypothesis that CHP inhibits SC differentiation by specifically inhibiting the secretion of collagen, cultures grown in the presence or absence of CHP were metabolically labeled with [3H]leucine and the media were analyzed for relative amounts of (a) collagenous and noncollagenous proteins by assay with bacterial collagenase and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), or (b) triple-helical collagen by pepsin digestion followed by SDS-PAGE. The results indicate that although CHP inhibited the accumulation of secreted collagen in the culture medium and disrupted collagen triple-helix formation, it also significantly inhibited the accumulation of secreted noncollagenous proteins in the medium. CHP had no significant effect on either total protein synthesis (medium plus cell layer) or cell number. We conclude that CHP does not act as a specific inhibitor of collagen secretion in this system, and thus data from these experiments cannot be used to relate SC collagen production to other aspects of SC differentiation. We discuss the evidence for and against specificity of CHP action in other systems.     

Fetal bovine bone cells synthesize bone-specific matrix proteins

We isolated cells from both calvaria and the outer cortices of long bones from 3- to 5-mo bovine fetuses. The cells were identified as functional osteoblasts by indirect immunofluorescence using antibodies against three bone-specific, noncollagenous matrix proteins (osteonectin, the bone proteoglycan, and the bone sialoprotein) and against type 1 collagen. In separate experiments, confluent cultures of the cells were radiolabeled and shown to synthesize and secrete osteonectin, the bone proteoglycan and the bone sialoprotein by immunoprecipitation and fluorography of SDS polyacrylamide gels. Analysis of the radiolabeled collagens synthesized by the cultures showed that they produced predominantly (approximately 94%) type I collagen, with small amounts of types III and V collagens. In agreement with previous investigators who have employed the rodent bone cell system, we confirmed in bovine bone cells that (a) there was a typical cyclic AMP response to parathyroid hormone, (b) freshly isolated cells possessed high levels of alkaline phosphatase, which diminished during culture but returned to normal levels in mineralizing cultures, and (c) cells grown in the presence of ascorbic acid and beta-glycerophosphate rapidly produced and mineralized an extracellular matrix containing largely type I collagen. These results show that antibodies directed against bone-specific, noncollagenous proteins can be used to clearly identify bone cells in vitro.     

Alteration of collagenous protein profile in congestive heart failure secondary to myocardial infarction

The rat model of myocardial infarction is characterized by progressive cardiac hypertrophy and failure. Rats with infarcts greater than 30% of the left ventricle exhibited early and moderate, stages of heart failure 4 and 8 weeks after the occlusion of the left coronary artery, respectively. As heart failure is usually associated with remodeling of the extracellular matrix, a histological and biochemical study of cardiac collagenous proteins was carried out using failing hearts. Total collagen content in the right ventricle increased at 2, 4, and 8 weeks following occlusion of the left coronary artery whereas such a change in viable left ventricle was seen after 4 and 8 weeks. Total cardiac hydroxyproline concentration was increased in both right and left ventricular samples from the infarcted animals when compared to those of control; this increase was due to elevation of pepsin-insoluble collagen fraction. The myocardial noncollagenous/collagenous protein ratio was decreased in experimental right and left ventricular samples when compared to control samples. These findings suggest that an increase in cross-linking of cardiac collagen as well as disparate synthesis of collagenous and noncollagenous proteins occurs in this model of congestive heart, failure.     

牙本质基质蛋白1及其对骨形成的调控作用

牙本质基质蛋白1(dentin matrix protein 1,DMP1)是一种高度磷酸化的偏酸性非胶原蛋白, 属于小整合素结合配体N端连接糖蛋白（small integrin-binding ligand, N-linked glycoprotein, SIBLINGs）家族.和SIBLINGs家族其它成员一样,DMP1基因定位于人类染色体4q21除存在于牙组织外,该蛋白还普遍分布于骨组织中.在骨组织与细胞中已发现4种DMP1的主要存在形式,即全长DMP1、57 kD C-DMP1、37 kD N-DMP1、DMP1-PG.它们的分布与功能均不相同,但对骨的正常形成均有重要意义. DMP1的氨基酸序列拥有大量的酸性结构域,携带负电荷,与钙离子有较强的结合能力.它在体外能够促进羟基磷灰石形成,并调控细胞分化,在体内参与硬组织的矿化过程.另外,DMP1的水解过程对其调控矿化的功能十分关键.人体内DMP1基因的突变可导致常染色体隐性低血磷性佝偻病.本文就近几年对DMP1基因结构与调控、蛋白结构与代谢、在骨组织与细胞中的分布及其对骨形成调控作用的研究进展作一综述.     

Effects of cis-4-hydroxy- -proline, an inhibitor of Schwann cell differentiation, on the secretion of collagenous and noncollagenous proteins by Schwann cells

The proline analog cis-4-hydroxy- -proline (CHP) was previously shown to inhibit both Schwann cell (SC) differentiation and extracellular matrix (ECM) formation in cultures of rat SCs and dorsal root ganglion neurons. We confirmed that CHP inhibits basal lamina formation by immunofluorescence with antibodies to laminin, type IV collagen, and heparan sulfate proteoglycan. In order to test the hypothesis that CHP inhibits SC differentiation by specifically inhibiting the secretion of collagen, cultures grown in the presence or absence of CHP were metabolically labeled with [³H]leucine and the media were analyzed for relative amounts of (a) collagenous and noncollagenous proteins by assay with bacterial collagenase and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), or (b) triple-helical collagen by pepsin digestion followed by SDS-PAGE. The results indicate that although CHP inhibited the accumulation of secreted collagen in the culture medium and disrupted collagen triple-helix formation, it also significantly inhibited the accumulation of secreted noncollagenous proteins in the medium. CHP had no significant effect on either total protein synthesis (medium plus cell layer) or cell number. We conclude that CHP does not act as a specific inhibitor of collagen secretion in this system, and thus data from these experiments cannot be used to relate SC collagen production to other aspects of SC differentiation. We discuss the evidence for and against specificity of CHP action in other systems.     

Distribution of bone inductive proteins in mineralized and demineralized extracellular matrix

Implantation of demineralized extracellular bone matrix results in new bone formation locally. Although the precise molecular mechanisms are not known, the reconstitution of matrix proteins less than 50,000 daltons with collagenous residue results in bone induction. The aim of the present investigation was to ascertain the distribution of the bone inductive protein(s) in various compartments of the tissue. A sequential extraction of mineralized bone matrix was employed: (1) 4 M guanidine HCl to extract proteins that are cell associated and not masked by mineral; (2) 0.5 M EDTA to dissolve the mineral phase; (3) 4 M guanidine HCl to reextract the collagenous matrix-associated proteins under dissociative conditions; (4) 4 M guanidine HCl containing 0.5 M EDTA to release any other residual proteins. This sequential method revealed that about 25% of total biological activity of bone induction is associated with first guanidine extraction, about 15% with the mineral phase and the rest of the activity is tightly associated with the collagenous matrix.     

A transmission electron microscope study using vitrified ice sections of predentin: structural changes in the dentin collagenous matrix prior to mineralization

The assembly of the collagenous organic matrix prior to mineralization is a key step in the formation of bones and teeth. This process was studied in the predentin of continuously forming rat incisors, using unstained vitrified ice sections examined in the transmission electron microscope. Progressing from the odontoblast surface to the mineralization front, the collagen fibrils thicken to ultimately form a dense network, and their repeat D-spacings and banding patterns vary. Using immunolocalization, the most abundant noncollagenous protein in dentin, phosphophoryn, was mapped to the boundaries between the gap and overlap zones along the fibrils nearest the mineralization front. It thus appears that the premineralized collagen matrix undergoes dynamic changes in its structure. These may be mediated by the addition and interaction with the highly anionic noncollagenous proteins associated with collagen. These changes presumably create a collagenous framework that is able to mineralize.     

Comprehensive analysis of the extracellular proteins from Xanthomonas campestris pv. campestris B100

The extracellular proteome of Xanthomonas campestris pv. campestris (Xcc) cultivated in minimal medium was isolated from the cell-free culture supernatant and separated by two-dimensional gel electrophoresis. This technique resolved 97 clearly visible protein spots, which were excised, digested with trypsin and identified on the basis of their peptide mass fingerprints generated by matrix assisted laser desorption/ionisation-time of flight-mass spectrometry. Using this approach 87 different proteins could be distinguished. The Signal P software predicted putative signal peptides for 53% of the extracellular proteins. These proteins are probably transported over the inner membrane and are localized in the periplasm, the outer membrane or secreted into the extracellular space. Among the secreted proteins are 11 degradative enzymes, which are involved in pathogenesis of Xcc. The proteins without obvious secretion signals are known to serve functions in the cytosol. How the cytosolic proteins are delivered to the extracellular space remains unclear.     

On the role of type IX collagen in the extracellular matrix of cartilage: type IX collagen is localized to intersections of collagen fibrils

The tissue distribution of type II and type IX collagen in 17-d-old chicken embryo was studied by immunofluorescence using polyclonal antibodies against type II collagen and a peptic fragment of type IX collagen (HMW), respectively. Both proteins were found only in cartilage where they were co-distributed. They occurred uniformly throughout the extracellular matrix, i.e., without distinction between pericellular, territorial, and interterritorial matrices. Tissues that undergo endochondral bone formation contained type IX collagen, whereas periosteal and membranous bones were negative. The thin collagenous fibrils in cartilage consisted of type II collagen as determined by immunoelectron microscopy. Type IX collagen was associated with the fibrils but essentially was restricted to intersections of the fibrils. These observations suggested that type IX collagen contributes to the stabilization of the network of thin fibers of the extracellular matrix of cartilage by interactions of its triple helical domains with several fibrils at or close to their intersections.     

Encapsulation of foreign particles in vitro by separated blood cells from crayfish,Astacus leptodactylus

Summary Bone matrix consists of type-I collagen and noncollagenous proteins. The latter represent only 10% of its total protein content. Since type-I collagen is also present in various other connective tissue sites (e.g., skin) it cannot be considered as bone specific. Among the non-collagenous components osteonectin — a 32 kilodalton (KD) glycoprotein linking mineral to collagen fibrils — is thought to be bone specific due to its biochemical properties. In the present study various skeletal and non-skeletal tissues were investigated for the presence of osteonectin by means of immunocytochemical methods. Two polyclonal antibodies against human and bovine osteonectin were applied. Immunocytochemically, osteonectin could be demonstrated in active osteoblasts and osteoprogenitor cells as well as in young osteocytes, while aged, quiescent osteocytes did not contain the protein, suggesting that the protein is a marker of the osteoblastic functional differentiation of bone cells. Osteonectin was absent in all non-skeletal tissues with the exception of chondrocytes in so-called mineralizing chondroid bone.     

Cystein-proteinase localization in osteoclasts: An immunocytochemical study

Summary Cysteine-proteinases such as cathepsin B and G were localized in rat osteoclasts, by an indirect protein A-immunogold labeling technique, on post-embedded ultrathin sections. In osteoclasts, specific immunogold labeling of both anti-cathepsin B and G was localized in Golgi vesicles, lysosomes, pale vacuoles of various sizes, and the extracellular canals of ruffled borders; no immunoreactivity was seen in the cytoplasmic matrix, mitchondria, cisterns of the rough endoplasmic reticulum, or nuclei. The presence of immunolabeling of cathepsins in osteoclasts and in the subosteoclastic compartment suggests that these enzymes are involved in the extracellular degradation of collagen and other noncollagenous bone matrix proteins.     

Specific colorimetric detection of o-diphenols and 3,4-dihydroxyphenylalanine-containing peptides

Interpretation of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis results for polypeptides which contain both collagenous and noncollagenous regions may be somewhat ambiguous since collagenous chains obey a different molecular weight vs mobility relationship than reduced globular proteins. In a recent study [Freytag, J. W., Noelken, M. E., and Hudson, B. G., 1979, Biochemistry18, 4761–4768], however, it was found that the α chains of calf skin collagen obeyed the same size-mobility relationship as reduced globular proteins when the number of residues was used as a measure of size. We extended that study over a broad size range and found the same result for 581 to 2104 residue polypeptides when 5% gels were used, and for 217 to 1052 residue polypeptides with 9% gels. On the other hand, SDS complexes of collagenous chains having fewer than 300 residues migrated considerably more slowly through 12.5% gels than their counter-parts from globular proteins. Also, SDS complexes of α_s1-, β-, and γ₂-casein which have 8.5, 16.7, and 20 mol% proline, respectively, had mobilities between those of SDS complexes of collagenous polypeptides and their reduced globular protein counterparts with the same number of residues. Our results indicate that SDS-polyacrylamide electrophoresis can be used to determine accurately the number of residues of collagenous polypeptides in the 217 to 2104 residue size range if appropriate gel concentrations are used. However, this conclusion does not apply to high-proline polypeptides in general.     

Unraveling the human bone microenvironment beyond the classical extracellular matrix proteins: a human bone protein library

A characteristic feature of bone, differentiating it from other connective tissues, is the mineralized extracellular matrix (ECM). Mineral accounts for the majority of the bone tissue volume, being the remainder organic material mostly derived from collagen. This, and the fact that only a limited number of noncollagenous ECM proteins are described, provides a limited view of the bone tissue composition and bone metabolism, the more so considering the increasing understanding of ECM significance for cellular form and function. For this reason, we set out to analyze and extensively characterize the human bone proteome using large-scale mass spectrometry-based methods. Bone samples of four individuals were analyzed identifying 3038 unique proteins. A total of 1213 of these were present in at least 3 out of 4 bone samples. For quantification purposes, we were limited to noncollagenous proteins (NCPs) and we could quantify 1051 NCPs. Most classical bone matrix proteins mentioned in literature were detected but were not among the highly abundant ones. Gene ontology analyses identified high-abundance groups of proteins with a functional link to mineralization and mineral metabolism such as transporters, pyrophosphatase activity, and Ca(2+)-dependent phospholipid binding proteins. ECM proteins were as well overrepresented together with nucleosome and antioxidant activity proteins, which have not been extensively characterized as being important for bone. In conclusion, our data clearly demonstrates that human bone tissue is a reservoir of a wide variety of proteins. In addition to the classical osteoblast-derived ECM, we have identified many proteins from different sources and of unknown function in bone. Thus, this study represents an informative library of bone proteins forming a source for novel bone formation modulators as well as biomarkers for bone diseases such as osteoporosis.     

Intact growth factors are conserved in the extracellular matrix of ancient human bone and teeth: a storehouse for the study of human evolution in health and disease

For the first time we have extracted, solubilized and identified growth factors, such as insulin growth factor II (IGF-II), bone morphogenetic protein-2 (BMP-2), and transforming growth factor-beta (TGF-beta), from archaeological compact human bone and tooth dentin dating from the late pre-ceramic pottery Neolithic (late PPNB) and the early Middle Ages. These factors are typical of special physiological or pathological situations in the metabolism of bone. The extracellular matrix proteins from bone and teeth of individuals from the late PPNB and early Middle Ages were separated by 2-D electrophoresis and more than 300 different protein spots were detected by silver staining. The matrix protein patterns of compact bone and tooth from the same individual (early Middle Ages) are very different and only 16% of the protein spots were detected in both compact bone and tooth dentin.     

Nature and distribution of chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone

Summary The type and distribution of mineral binding and collagenous matrix-associated chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone were studied biochemically and immunocytochemically, using three monoclonal antibodies (mAb 2B6, 3B3, and 1B5). The antibodies specifically recognize oligosaccharide stubs that remain attached to the core protein after enzymatic digestion of proteoglycans and identify epitopes in chondroitin 4-sulphate and dermatan sulphate; chondroitin 6-sulphate and unsulphated chondroitin; and unsulphated chondroitin, respectively. In addition, mAb 2B6 detects chondroitin 4-sulphate with chondroitinase ACII pre-treatment, and dermatan sulphate with chondroitinase B pre-treatment. Bone proteins were extracted from fresh specimens with a three-step extraction procedure: 4m guanidine HCl (G-1 extract), 0.4m EDTA (E-extract), followed by guanidine HCl (G-2 extract), to characterize mineral binding and collagenous matrix associated proteoglycans in E- and G2-extracts, respectively. Biochemical results using Western blot analysis of SDS-polyacrylamide gel electrophoresis of E- and G2-extracts demonstrated that mineral binding proteoglycans contain chondroitin 4-sulphate, chondroitin 6-sulphate, and dermatan sulphate, whereas collagenous matrix associated proteoglycans showed a predominance of dermatan sulphate with a trace of chondroitin 4-sulphate and no detectable chondroitin 6-sulphate or unsulphated chondroitin. Immunocytochemistry showed that staining associated with the mineral phase was limited to the walls of osteocytic lacunae and bone canaliculi, whereas staining associated with the matrix phase was seen on and between collagen fibrils in the remainder of the bone matrix. These results indicate that mineral binding proteoglycans having chondroitin 4-sulphate, dermatan sulphate, and chondroitin 6-sulphate were localized preferentially in the walls of the lacunocanalicular system, whereas collagenous associated dermatan sulphate proteoglycans were distributed over the remainder of the bone matrix.     

Dentin matrix protein 1 immobilized on type I collagen fibrils facilitates apatite deposition in vitro

During bone and dentin mineralization, the crystal nucleation and growth processes are considered to be matrix regulated. Osteoblasts and odontoblasts synthesize a polymeric collagenous matrix, which forms a template for apatite initiation and elongation. Coordinated and controlled reaction between type I collagen and bone/dentin-specific noncollagenous proteins are necessary for well defined biogenic crystal formation. However, the process by which collagen surfaces become mineralized is not understood. Dentin matrix protein 1 (DMP1) is an acidic noncollagenous protein expressed during the initial stages of mineralized matrix formation in bone and dentin. Here we show that DMP1 bound specifically to type I collagen, with the binding region located at the N-telopeptide region of type I collagen. Peptide mapping identified two acidic clusters in DMP1 responsible for interacting with type I collagen. The collagen binding property of these domains was further confirmed by site-directed mutagenesis. Transmission electron microscopy analyses have localized DMP1 in the gap region of the collagen fibrils. Fibrillogenesis assays further demonstrated that DMP1 accelerated the assembly of the collagen fibrils in vitro and also increased the diameter of the reconstituted collagen fibrils. In vitro mineralization studies in the presence of calcium and phosphate ions demonstrated apatite deposition only at the collagen-bound DMP1 sites. Thus specific binding of DMP1 and possibly other noncollagenous proteins on the collagen fibril might be a key step in collagen matrix organization and mineralization.     

Noncollagenous bone proteins in experimental rickets in the rat

Rats were raised in the absence of vitamin D in utero and throughout post-fetal life and neither 1,25-dihydroxyvitamin D3 nor related metabolites were detected in serums. No changes were observed in the relative amount of extractable noncollagenous bone proteins (NCP) in rachitic compared to vitamin-D-repleted animals. As expected, the relative levels of the mineral-bound, serum-derived albumin and 2-HS glycoprotein were unaffected in bones of rachitic animals. Interestingly, the vitamin D deficiency also did not have dramatic effects on several bone cell-derived noncollagenous proteins including: bone proteoglycans I & 11, bone sialoprotein li osteonectin, and osteocalcin. In contrast to the proteoglycans, the bone sialoprotein II and osteonectin were found in the nonmineral compartment of the rachitic animals, presumably bound to the wide osteoid seam.     

Primary structure and phosphorylation of dentin matrix protein 1 (DMP1) and dentin phosphophoryn (DPP) uniquely determine their role in biomineralization

The SIBLING (small integrin-binding ligand N-linked glycoproteins) family is the major group of noncollagenous proteins in bone and dentin. These extremely acidic and highly phosphorylated extracellular proteins play critical roles in the formation of collagenous mineralized tissues. Whereas the lack of individual SIBLINGs causes significant mineralization defects in vivo, none of them led to a complete cessation of mineralization suggesting that these proteins have overlapping functions. To assess whether different SIBLINGs regulate biomineralization in a similar manner and how phosphorylation impacts their activity, we studied the effects of two SIBLINGs, dentin matrix protein 1 (DMP1) and dentin phosphophoryn (DPP), on mineral morphology and organization in vitro. Our results demonstrate distinct differences in the effects of these proteins on mineralization. We show that phosphorylation has a profound effect on the regulation of mineralization by both proteins. Specifically, both phosphorylated proteins facilitated organized mineralization of collagen fibrils and phosphorylated DMP1-induced formation of organized mineral bundles in the absence of collagen. In summary, these results indicate that the primary structure and phosphorylation uniquely determine functions of individual SIBLINGs in regulation of mineral morphology and organization.