Non-collagenous proteins of rat dentin. Evidence that phosphoprotein is not covalently bound to collagen

The non-collagenous proteins of rat dentin that remain firmly bound to the matrix after demineralization were studied in order to ascertain if they are covalently linked to insoluble dentin collagen. After solubilization with CNBr or with bacterial collagenase, unusually small amounts of dentin phosphoprotein were detected in the matrix. The phosphoprotein obtained by CNBr digestion of the matrix was separated from collagen peptides using two chromatographic steps. Thus even this small quantity of phosphoprotein found in decalcified rat dentin matrix was not covalently bound to collagen.     

Participation of hepatocytes in the production of basement membrane components in human and rat liver during the perinatal period

Little is known about the role of the extracellular matrix in cellular growth, migration and differentiation in the developing liver. The distribution and origin of the main constituents of the hepatic extracellular matrix have never been studied during liver differentiation. We have investigated the extracellular and intracellular distribution of fibronectin, laminin and types I, III and IV collagen in both rat and human liver during the perinatal period by light and electron microscopy, using the indirect immunoperoxidase method. All these components were demonstrated extracellularly, located mainly in portal spaces and, to a lesser extent, surrounding central veins. In perisinusoidal spaces, variations in distribution were observed depending on the matrix protein, the age of the donor and the species. In fetal rat liver, fibronectin formed a continuous layer around hepatocyte clusters while laminin and type III procollagen were present in small amounts. Collagens and laminin were visualized more easily in newborn rat liver. Fetal and newborn human liver contained higher amounts of matrix components than their rat counterparts. Fibronectin also reacted strongly in the sinusoid, and laminin and collagens formed discontinuous deposits. The source of this extracellular matrix was demonstrated to be of mixed origin. The major finding was the presence of immunoreactive laminin in the rough endoplasmic reticulum of hepatocytes irrespective of the age or species. In addition, hepatocytes contained large amounts of fibronectin and little of type I collagen. Another basement membrane component, type IV collagen, was also found in hepatocytes from all groups except fetal rat. Perisinusoidal cells also contained various matrix components including laminin, type III procollagen and, again with the exception of fetal rat liver, type IV collagen. The greater amounts of basement membrane components in the sinusoids of developing liver than in adult tissue and the participation of immature hepatocytes in the production of laminin and to a lesser degree of type IV collagen suggest that these matrix proteins play a critical role during liver differentiation.     

Random distribution of mammalian replication origins in matrix and total nuclear DNA

Nuclear matrices from mouse and rat tumour cells were isolated and characterized by their microscopic appearance, protein profiles and DNA content. They presented well-defined structures containing 15-20% of the nuclear protein and 1-3% of the nuclear DNA. Matrix DNAs were immobilized on nitrocellulose filters and hybridized to nick-translation 32P-labelled homologous DNA fragments containing the corresponding replication origins. As control total nuclear DNAs were also immobilized on filters and hybridized to origin-containing DNAs. The origin-containing DNAs hybridized to the same extent to both matrix and total DNAs, which showed that they contained the same proportion of origin sequences. In an alternative series of experiments, plasmids containing either rat or mouse replication origins were immobilized on filters and were hybridized with in vitro 32P-labelled matrix and total nuclear DNAs. Here again both matrix and total nuclear DNAs hybridized to the same extent with the origin-carrying plasmids, which showed that neither rat nor mouse matrix DNAs were enriched in DNA replication origin sequences.     

Characterization of lamin proteins in BHK cells

Lamins are structural proteins found in rat liver nuclear envelope and are major constituents of the nuclear matrix. 2-D gel electrophoresis indicates that BHK cell nuclear matrix is composed of four major proteins (62 kD, 68 kD, 70 kD and 72 kD). Three of these proteins are very similar to lamins A, B and C of rat liver nuclear envelope according to their molecular mass and isoelectric points. An anti-serum specific to BHK matrix proteins has been raised. On 2-D immunoblot, this serum detects all the 62, 68 and 72 kD polypeptide isovariants but only one of the two isovariants of the 70 kD polypeptide. Rat lamins A, B and C react with the anti-BHK matrix serum. However, when a monoclonal antibody to rat liver lamins A, B and C is used (Burke, B, Tooze, J & Warren, G, EMBO j 2 (1983) 361 [23]), only the 72 kD (lamin A-like) and the 62 kD (lamin C-like) BHK polypeptides are detected. Our results suggest that although a strong similarity exists between BHK and rat lamins, there is no identical cross-reactivity between the two species.     

Rat chromosome 5 (q22–23) contains elements that control cell morphology and interactions with the extracellular matrix: A study of normal fibroblast X malignant hepatoma cell hybrids

Cell interactions with the extracellular matrix are consistently modified in neoplasia. Malignant transformation has been correlated with modifications in the synthesis and distribution of matrix components and with alterations of cell adhesive properties to these components. A particular class of genes, able to suppress the transformed phenotype in normal cells, may be involved in those phenotypic changes. By studying somatic cell hybrids between mouse hepatoma (BWTG3) cells and normal rat skin fibroblasts (RSF), Islam and co-workers were able to localize a gene or a group of genes controlling anchorage dependence and cell growth in vitro. This (or these) gene(s) was (were) assigned to the q22-23 fragment of rat chromosome 5. In the present study, we compare the morphology and the interactions with the extracellular matrix proteins (laminin, fibronectin, and collagen IV) and the synthesis of these proteins by RSF X BWTG3 hybrid cells that had either retained (BS181p10) or lost (BS181a5) the q22-23 region of rat chromosome 5. Our results suggest that the rat 5q22-23 fragment controls a part of the cell differentiation program including morphology, attachment to extracellular matrix, and synthesis of some matrix proteins, particularly alpha 1 and alpha 2 chains of collagen IV.     

Electron microscopic study of condylar cartilage of rat mandible stained with ruthenium red: proteoglycans and hypertrophic chondrocytes

Ultrastructure of hypertrophic chondrocytes and extracellular matrix in condylar cartilage of rat mandible was studied in conjunction with ruthenium red staining. Special care was given to the preservation of proteoglycans in the extracellular matrix. Ruthenium red-positive granules were observed in the pericellular matrix of condylar chondrocytes, and their size and number increased around the hypertrophic cells. However, these granules disappeared in the lowest hypertrophic zone, in which uncalcified cartilage matrix was also disintegrated prior to initiation of ossification. Moreover, hypertrophic chondrocytes observed at the lowest zone appeared intact in their ultrastructural features, i.e., containing numbers of lysosomes and coated vesicles in the cytoplasm facing the blood capillaries. The results strongly suggest that the lowest hypertrophic chondrocytes in rat condylar cartilage may have an active role in the degradation and resorption of the pericellular matrix, especially proteoglycans, and uncalcified matrix, which changes seem an essential step for the initiation of endochondral ossification.     

Glycosaminoglycan synthesis by rat fibroblast monolayers: effects of serum and solubilized bone matrix fractions

Glycosaminoglycan synthesis by fibroblasts, from skeletal muscle of the neonatal rat, is stimulated by a bone matrix preparation, soluble in isotonic medium, obtained by extracting decalcified rat bone with 4 M guanidine HCl. The stimulation in glycosaminoglycan synthesis is dependent on the presence of serum in the culture, but the stimulatory effect can be clearly distinguished from that of serum. The stimulatory activity in the bone matrix has been fractionated by ion-exchange chromatography and coelutes largely with anionic, non-collagenous matrix glycoproteins.     

Morphogenetic Effects of Soluble Laminin-5 on Cultured Epithelial Cells and Tissue Explants

The rat cell line 804G assembles an extracellular matrix which induces not only the rapid adhesion and spreading of epithelial cells but also the assembly of a cell–matrix attachment device called the hemidesmosome. The major component of this matrix is laminin-5. We have purified rat laminin-5 from medium conditioned by 804G cells. Epithelial cells which are co-incubated with medium supplemented with soluble laminin-5 adhere and spread rapidly. Furthermore, human carcinoma cells undergo a dramatic morphologic change in the presence of laminin-5 and form orderly arrays resembling epithelial sheets. Soluble rat laminin-5 is selectively incorporated into an insoluble matrix of epithelial cellsin vitro,since rat-specific laminin-5 antibodies stain cell–substrate contacts. Addition of medium containing soluble laminin-5 to explanted, human corneal rims induces assembly of hemidesmosomes, important cell–matrix attachment devices. Furthermore, rat-specific laminin-5 antibodies stain areas of contact between corneal epithelium and basement membrane, indicating that rat laminin-5 from the medium is incorporated into basement membrane. We discuss the use of laminin-5 as a medium supplement for the culture of both epithelial cells and epithelial tissue explants.     

Calreticulin--an endoplasmic reticulum protein with calcium-binding activity is also found in the extracellular matrix.

Previous studies have reported that calreticulin (CRT), a calcium-binding and chaperoning protein, is expressed only in the endoplasmatic reticulum, nucleus and at the cell surface. In this study we clearly show that odontoblasts and predentin matrix contain CRT. To our knowledge, this is the first time CRT has been described in the extracellular matrix. The expression of CRT was studied by immunohistochemistry, ultrastructural immunocytochemistry and in situ hybridization in developing rat teeth. CRT was detected as a 59-kDa protein in rat pulp cell culture medium and dentin extracellular matrix extract by Western blotting. The presence of the protein was shown in rat odontoblasts and predentin with immunohistochemistry. At the ultrastructural level, the labeling was distributed in the rat odontoblasts, ameloblasts and predentin. Northern blotting showed the presence of CRT mRNA in rat molars, which was confirmed by in situ hybridization in odontoblasts and ameloblasts. We now present the first convincing evidence that CRT is found in extracellular matrix where it may play an important role in mineralization.     

Cell-free translation of mitochondrial nicotinamide nucleotide transhydrogenase

Mammalian nicotinamide nucleotide transhydrogenase is translated as a 5000 daltons larger molecular weight precursor in a cell-free system programmed with rat liver polysomes. The mature rat liver enzyme had the same molecular weight as the purified beef heart enzyme, 115 000 daltons. The precursor was not processed in vitro by liver mitochondria or by a rat liver mitochondrial matrix fraction, nor did it appear to bind to mitochondria. In contrast, pre-FeS protein of the cytochrome bc₁ complex was processed in the same samples by both mitochondria and matrix, suggesting an important difference in the processing mechanisms or in the efficiency of processing of the two precursors.     

The effect of extracellular matrix interactions on morphologic transformation in vitro

There is emerging evidence that the structure and function of a cell is dependent in part on the contacts that cells make with the extracellular matrix. We report here the effect of extracellular matrices secreted from both normal and tumor cells have on the structure of normal rat kidney epithelial cells. Normal rat kidney cells plated on the basement membrane secreted by tumor cells adopt a morphology and phenotype which closely resembles a Kirsten-ras transformed normal rat kidney cell. This morphologic transformation was not observed for cells plated on individual extracellular matrix components or on basement membrane secreted by normal placenta cells. This suggests that tumor derived basement membrane has unique characteristics which may cause morphologic transformation of normal rat kidney cells.     

Comparison of bone inductive proteins of rat and porcine bone matrix

Subcutaneous implantation of demineralized bone matrix in allogenic rats induces a sequence of events resulting in de novo formation of cartilage, bone and bone marrow. In the present study endochondral bone formation by demineralized porcine matrix was studied and compared with the rat bone matrix. Endochondral bone formation was induced by 4M guanidine hydrochloride fraction IV (less than 50,000 daltons) of Sepharose CL-6B gel filtration but not by whole extract or by demineralized porcine bone matrix. Sephacryl S-200 gel filtration of the osteoinductive proteins of fraction IV showed the Porcine osteoinductive factor to be associated with protein fraction III (less than 20,000 daltons) whereas the rat with fraction II (between 20,000 and 30,000 daltons) of the chromatographic profile indicating an apparent difference in molecular weight of the osteoinductive factors between these two species.     

Dual role for the latent transforming growth factor-beta binding protein in storage of latent TGF-beta in the extracellular matrix and as a structural matrix protein

The role of the latent TGF-beta binding protein (LTBP) is unclear. In cultures of fetal rat calvarial cells, which form mineralized bonelike nodules, both LTBP and the TGF-beta 1 precursor localized to large fibrillar structures in the extracellular matrix. The appearance of these fibrillar structures preceded the appearance of type I collagen fibers. Plasmin treatment abolished the fibrillar staining pattern for LTBP and released a complex containing both LTBP and TGF-beta. Antibodies and antisense oligonucleotides against LTBP inhibited the formation of mineralized bonelike nodules in long-term fetal rat calvarial cultures. Immunohistochemistry of fetal and adult rat bone confirmed a fibrillar staining pattern for LTBP in vivo. These findings, together with the known homology of LTBP to the fibrillin family of proteins, suggest a novel function for LTBP, in addition to its role in matrix storage of latent TGF-beta, as a structural matrix protein that may play a role in bone formation.     

Isolation of glyoxalase II from two different compartments of rat liver mitochondria. Kinetic and immunochemical characterization of the enzymes

Two separate pools of glyoxalase II were demonstrated in rat liver mitochondria, one in the intermembrane space and the other in the matrix. The enzyme was purified from both sources by affinity chromatography on S-(carbobenzoxy)glutathione-Affi-Gel 40. From both crude and purified preparations polyacrylamide gel-electrophoresis resolved multiple forms of glyoxalase II, two from the intermembrane space and five from the matrix. Among the thioesters of glutathione tested as substrates, S-D-lactoylglutathione was hydrolyzed most efficiently by the enzymes from both sources. Significant differences were observed in the specificities between the intermembrane space and matrix enzymes with S-acetoacetylglutathione, S-acetylglutathione, S-propionylglutathione and S-succinylglutathione as substrates. Pure glyoxalase II from rat liver cytosol was chemically polymerized and used as antigen. Antibodies were raised in rabbits and the antiserum was used for comparison of the two purified mitochondrial enzymes with cytosolic glyoxalase II by immunoblotting. The enzyme purified from the intermembrane space cross-reacted with the antiserum, but the matrix glyoxalase II did not. The results give evidence for the presence in rat liver mitochondria of two species of glyoxalase II with differing characteristics. Only the enzyme from the intermembrane space appears to resemble the cytosolic glyoxalase II forms.     

The nuclear protein p30 specifically interacts with a nuclear matrix attachment region from the rat genome

In our previous study, a 454 bp DNA fragment was isolated from rat genomic DNA as an element which interacts with nuclear matrix proteins, i.e. a Matrix Associated Region (MAR). Computer analyses revealed that the right half of this fragment, named RME (Rat MAR Element), possesses a high matrix association potential and is likely to be responsible for the matrix association of the whole sequence. RME was used as a probe in an electrophoretic mobility shift assay (EMSA), and with the use of Southwestern blotting, a rat liver nuclear protein which binds specifically to it was identified. Its molecular mass was estimated by SDS-PAGE as 30 kDa (p30). Polyclonal antibodies raised against protein-RME complexes caused a super-shift of specific complexes in EMSA, and bound to p30 in nuclear extracts of rat liver in Western blotting. The immunofluorescence labelling of a rat embryonic fibroblast cell monolayer with anti-p30 antibody revealed a mainly intranuclear pattern of staining.     

Protease activity of the nuclear matrix of rat hepatocytes

It was demonstrated that the nuclear matrix of rat liver possesses the protease activity. The specific activity of nuclear matrix proteases exceeds that of intact nuclei 7-fold. The optimum activity of nuclear matrix proteases is observed at pH 8-9. The protease activity of the nuclear matrix is inhibited by p-chloromercuribenzoate, N-ethylmaleimide, EDTA, phenylmethylsulfonyl fluoride. This suggests that thiol, serine and metalloproteases are associated with the nuclear matrix.     

Identification of a meiotic prophase-specific nuclear matrix protein in the rat

We have identified a 110-kDa pI 5.6 phosphoprotein with DNA binding properties in the rat pachytene spermatocyte nuclear matrix. By immunoblotting and indirect immunofluorescence assays using polyclonal antibodies against the 110-kDa protein, we observed that it was germ cell nuclear matrix specific, more prominent in pachytene spermatocytes compared to premeiotic spermatogonia or postmeiotic round spermatids, and present in rat oocytes and in germ cells of mouse and monkey. We propose that this protein could play an important role in the meiotic process.