The importance of the generalist.
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Family Practice↗
Biomedical subjects
C W Prince
Publications and source records attributed to C W Prince.
Osteopontin, a substrate for transglutaminase and factor XIII activity.
Osteopontin (OPN), an extracellular matrix cell adhesion protein, was found to serve as a substrate for the incorporation of radiolabelled putrescine mediated by a commercial preparation of guinea pig liver transglutaminase. Preliminary evidence also suggests that OPN serves as a substrate for the plasma transglutaminase, Factor XIIIa. While the protein substrates to which OPN is linked in vivo have not been identified, it is reasonable to speculate that this capacity of OPN may dictate its extracellular location and thereby affect its role in bone homeostasis, tumorigenesis, metastasis, resistance to bacterial infections or, perhaps, wound repair.
Amino Acid Sequence↗
1 alpha,25-dihydroxyvitamin D3 stimulates synthesis and secretion of nonphosphorylated osteopontin (secreted phosphoprotein 1) in mouse JB6 epidermal cells.
Murine JB6 epidermal cells can be irreversibly transformed into tumorigenic cells by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate. One feature of this transformation is induction of the synthesis and secretion of the phosphoprotein osteopontin (also called secreted phosphoprotein 1 and previously referred to as transformation-related phosphoprotein, 2ar, bone sialoprotein 1, or Mr 44,000 bone phosphoprotein), an arginylglycylaspartic acid-containing cell adhesion glycoprotein the expression of which has been implicated in tumorigenesis and metastasis. Since 1 alpha,25-dihydroxyvitamin D3, calcitriol, also transforms JB6 cells and, in other cell types, regulates osteopontin synthesis, we hypothesized that calcitriol-mediated transformation of JB6 cells would also cause induction of osteopontin synthesis and secretion. Metabolic labeling with 32PO4 of near confluent JB6 cells (clone 41.5a) treated with calcitriol (0.1-100 ng/ml) for up to 48 h revealed only a minimal production of osteopontin, which is the major phosphoprotein secreted by 12-O-tetradecanoylphorbol-13-acetate-treated cells. Similar treatment followed by labeling with [35S]methionine showed a substantial dose-dependent increase in the synthesis and secretion of osteopontin. This induction was not associated with increased cell proliferation or with cell transformation, as assayed by anchorage-independent growth. Calcitriol-treated cells were morphologically indistinguishable from control cells, while 12-O-tetradecanoylphorbol-13-acetate-treated cells acquired a distinctive morphology. No induction of osteopontin was found with 25-hydroxyvitamin D3 or 24R,25-dihydroxyvitamin D3. These results show that calcitriol induces the synthesis and secretion of a nonphosphorylated form of osteopontin, in a cell type which normally makes little or none of this protein, and that the induction is not correlated with the tumorigenic transformation of these cells.
Animals↗
Cell attachment activity of the 44 kilodalton bone phosphoprotein is not restricted to bone cells.
Proteins that promote cell migration, attachment and spreading are considered to play an important role in the regulation of cell function. Recently, a 44 kilodalton bone phosphoprotein (44K BPP) was shown to enhance the attachment of gingival fibroblasts and osteoblasts in vitro. The potential importance of this attachment protein in the regulation of mineralized tissue homeostasis prompted us to evaluate its ability to promote the attachment and migration of several other cell types. All the fibroblast cell lines and non-transformed calvaria cell lines assayed exhibited enhanced attachment and spreading in response to 44K BPP. Rat osteosarcoma cells (ROS 17/2.8) expressing osteoblast-like features, exhibited enhanced attachment in response to 44K BPP, while non-osteoblast-like cells (ROS 25/1) obtained from the same osteosarcoma did not. Two epithelial cell lines, CCL4 and A431, demonstrated enhanced attachment when exposed to fibronectin or laminin, but not 44K BPP. Another epithelial-like cell line, HT 1080, derived from a fibrosarcoma, showed enhanced attachment in the presence of all three attachment proteins. Fibronectin, but not 44K BPP, promoted the chemotactic migration of fibroblasts. These studies indicate that the role of 44K BPP attachment protein in the regulation of cell behavior is not restricted to bone cells.
Animals↗
Secondary structure predictions for rat osteopontin.
Five computerized methods were used to predict the secondary structure of osteopontin - a bone-derived cell attachment protein. The amino terminal one-fifth and the carboxy terminal one-third of the 301 amino acid protein contain eight alpha helices (41% of the total residues). The middle of the molecule contains a very acidic region of no predicted structure followed by two segments of beta structure which flank the cell attachment site of osteopontin. Examination of the amino acid sequence also revealed a potential calcium binding loop and two potential heparin binding sites.
Amino Acid Sequence↗
Transcriptional regulation of osteopontin production in rat osteosarcoma cells by type beta transforming growth factor.
Type beta transforming growth factor (TGF beta) was shown to regulate the production of several extracellular matrix proteins. Osteopontin (OP) is a recently discovered bone matrix protein which was shown to promote the attachment of osteoblastic rat osteosarcoma ROS 17/2.8 cells to their substrate. We examined the effects of TGF beta on OP production and OP mRNA in ROS 17/2.8 cells. Four-day treatment with 4 ng/ml TGF beta 1 increased substantially the level of osteopontin in the cell culture media, as estimated by immunoblotting. Metabolic labeling showed that this effect was associated with a 3-4-fold increase in OP biosynthesis. TGF beta 1 also increased, in a dose-dependent manner starting at 0.4 ng/ml, the steady-state level of OP mRNA. The increase in OP mRNA was first detected 48 h after the addition of TGF beta 1 and lasted at least until 120 h. The half-life of OP mRNA, estimated in the presence of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, was about 10 h and was not altered by TGF beta 1. On the other hand, the increase in OP mRNA was blocked by actinomycin D. Nuclear run-on assays indicated that TGF beta 1 increased the rate of transcription of the OP gene. Examination of hormonal interactions showed that TGF beta 1 opposed or compensated for the reduction in OP mRNA produced by dexamethasone and that TGF beta 1 did not further augment OP mRNA levels which had been increased by 1,25-dihydroxyvitamin D3 treatment. TGF beta 2 had similar effects on OP gene expression as TGF beta 1. In conclusion, TGF beta promotes the production of osteopontin in the osteoblastic osteosarcoma cells through a pathway which is at least in part mediated by transcriptional events.
Animals↗
44-kDal bone phosphoprotein (osteopontin) antigenicity at ectopic sites in newborn rats: kidney and nervous tissues.
Previous immunohistochemical data have shown that the 44-kDal bone phosphoprotein (44K BPP, also called sialoprotein I or oestopontin) recently isolated in our laboratory was synthesized by osteoblasts and osteocytes and was expressed early during differentiation of bone-forming cells. We report here the presence of 44K BPP antigenicity at certain ectopic sites, namely, the proximal-convoluted tubule of the kidney, neurons, sensory and secretory cells in the internal ear. To insure specificity and reproducibility, different immunohistochemical methods were used and affinity-purified antibodies against two separate preparations of pure 44K BPP were tested. In the cells of the proximal-convoluted tubule, 44K BPP immunoreactivity was observed within apical endocytotic vacuoles and within lysosomes. This staining thus correlates with the degradation of the 44K BPP epitope which we previously demonstrated to occur in serum. On the other hand, in the neurons of the acoustic ganglion and the sensory cells of the macula, 44K BPP immunoreactivity was associated with the Golgi apparatus indicating synthesis and secretion by these cells. The finding that the 44K BPP (or a structurally related molecule) is synthesized by neurons and neuroepithelial cells deserves further investigation with respect to a possible embryologic relationship between neuroectodermal cells and the precursors of some bone forming-cells of the skull.
Animals↗
Developmental expression of 44-kDa bone phosphoprotein (osteopontin) and bone gamma-carboxyglutamic acid (Gla)-containing protein (osteocalcin) in calcifying tissues of rat.
New aspects of the distribution and developmental appearance of the 44-kDa bone phosphoprotein (44K BPP, also called sialoprotein I or osteopontin) and bone gamma-carboxyglutamic acid (Gla)-containing protein (BGP, also called osteocalcin) during osteogenesis and dentinogenesis were investigated with immunocytochemical techniques using monospecific, affinity-purified polyclonal antibodies. Sections from newborn rat incisors and from various bone anlagen of newborn animals and fetuses were processed for detection of 44K BPP or BGP antigenicity. In addition, histochemical reactions for detection of alkaline phosphatase or calcium salts were performed on a number of the sections. The 44K BPP appears to be synthesized and secreted by chondrocytes only in the areas of cartilage-to-bone transition; these cells could participate indirectly in the process of bone formation by providing a suitable scaffold onto which primary marrow osteoblasts attach and spread. During osteogenesis, 44K BPP is found in bone-forming cells almost concomitantly with the appearance of alkaline phosphatase and before osteoid deposition, whereas BGP is still absent during early stages of mineralization. We hypothesize that this dramatic difference between the developmental appearance of 44K BPP and BGP reflects the delayed expression of the BGP gene relative to that of 44K BPP. In long-term cultures of bone marrow from adult mice, some fibroblastic cells expressed the 44K BPP phenotype; these cells could represent early osteogenic progenitor cells. Some experiments also suggested that, as with BGP, 44K BPP or an immunologically related protein is synthesized by some odontoblasts and secreted into predentin, prior to the onset of mineralization.
Alkaline Phosphatase↗
Isolation, characterization, and biosynthesis of a phosphorylated glycoprotein from rat bone.
A phosphorylated glycoprotein was purified from the mixture of proteins extracted by demineralization of rat bone with 0.5 M EDTA in 4 M guanidinium chloride. A high level of purity for the preparation was indicated by a single band on sodium dodecyl sulfate (SDS)-gradient gel electrophoresis, sedimentation equilibrium ultracentrifugal data, and by automated Edman degradation results. The molecular weight of the phosphoprotein was shown to be about 44,000 by sedimentation equilibrium analyses in 4 M guanidinium chloride, even though an Mr of 75,000 was obtained by 5-15% SDS-polyacrylamide gel electrophoresis. Subsequent analysis by 15% SDS-polyacrylamide gel electrophoresis gave an Mr of 45,000. Analytical data showed that the protein contained 16.6% carbohydrate, possibly including 1 N-linked oligosaccharide and 5-6 O-linked oligosaccharides. The aspartic acid- and glutamic acid-rich protein contained about 300 amino acid residues including 1 phosphothreonine and 12 phosphoserine residues. Alkaline beta-elimination/NaBH4 reduction data showed that the phosphate obtained by complete acid hydrolysis prior to amino acid analysis was equivalent to the phosphate subject to alkaline beta-elimination. In this experiment, the losses of serine plus threonine exceeded the amount of phosphate liberated by 5-6 residues/protein. These serine and threonine residues probably represent O-linked oligosaccharides, since the protein contained about this number of N-acetyl-galactosamine residues. That the phosphoprotein is synthesized and secreted by osteoblast-like cells was shown with cultures of clonal rat osteosarcoma cells. After pulsing with 32PO4 the proteins secreted into the medium were precipitated with trichloroacetic acid and the radiolabeled proteins were immunoadsorbed. A protein migrating in the same position, on 5-15% SDS-polyacrylamide gel electrophoresis (i.e. with an Mr = 75,000) and on 15% gels (Mr = 45,000), as the phosphoprotein obtained from bone could be specifically immunoprecipitated.
Amino Acids↗
Mechanism of fibroblast attachment to bone extracellular matrix: role of a 44 kilodalton bone phosphoprotein.
While the exact mechanisms regulating bone homeostasis are unknown, it is generally accepted that factors with the capacity to regulate cell attachment and spreading play a role in osteogenesis. A 44 kDa bone phosphoprotein (44K BPP), isolated from rat bone and synthesized by osteoblasts, was evaluated for its role in attachment and spreading of fibroblasts. In uncoated plates, enhanced cell attachment and spreading were observed when fibroblasts were exposed to the 44K BPP. The attachment properties of the bone phosphoprotein are different from those of fibronectin, in that the 44K BPP did not promote cell attachment in type I collagen wells, as was seen with fibronectin. Also, 44K BPP continued to enhance cell attachment up to 24 h, whereas cell attachment declined in time with cells exposed to fibronectin. Cycloheximide did not alter 44K BPP promotion of cell attachment, indicating that de novo protein synthesis was not required. These studies suggest that the 44K BPP is important in the regulation of cell attachment and spreading at sites of mineralization.
Bone and Bones↗
A comparative immunocytochemical study on the subcellular distributions of 44 kDa bone phosphoprotein and bone gamma-carboxyglutamic acid (Gla)-containing protein in osteoblasts.
Bone gamma-carboxyglutamic acid (Gla)-containing protein (BGP or osteocalcin) and 44 kDa bone phosphoprotein (44K BPP, also called Sialoprotein I or osteopontin) have been localized at the ultrastructural level in osteoblasts from woven bones of newborn rats. Frozen, undecalcified sections of periodate-lysine-paraformaldehyde fixed specimens were incubated with affinity purified, monospecific antibodies against BGP or 44K BPP. The sites of the antigen-antibody reaction were demonstrated by the avidin-biotin-peroxidase complex method using the Hanker-Yates reagent as a peroxidase substrate. In some cases immunostaining could only be achieved after detergent treatment. The immunostained sections were then flat-embedded in Epon 812 and processed for electron microscopy. Strong specific intracellular labeling was obtained with both antibodies, but the patterns of staining differed significantly: BGP antigenicity was mainly located in the endoplasmic reticulum (ER), whereas 44K BPP behaved as a Golgi-specific antigen. In both cases, however, we found no evidence for immunostained secretory vesicles. There was no correlation between the expression of BGP by osteoblasts and the morphological aspect of these cells, their apparent degree of polarization with respect to the bone matrix, or their relation with the mineralized phase.
Animals↗
Proteoglycans of mineralized matrices.
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Animals↗
1,25-Dihydroxyvitamin D3 regulates the biosynthesis of osteopontin, a bone-derived cell attachment protein, in clonal osteoblast-like osteosarcoma cells.
We investigated the effects of 1,25-dihydroxyvitamin D3 on the synthesis of osteopontin, a phosphorylated cell attachment glycoprotein, in ROS 17/2.8 cells, a clonal osteoblast-like rat osteosarcoma cell line. We observed a dose dependent increase in uptake of [32PO4] into osteopontin secreted into the medium. An increased incorporation of [35S]-methionine into secreted osteopontin suggested the effect was that of increased protein biosynthesis. Using a radioimmunoassay we demonstrated a dose dependent increase in the amount of secreted osteopontin, an increase which could be blocked by Actinomycin D, in response to 1,25-dihydroxyvitamin D3. These results suggest that the hormonal form of vitamin D regulates the biosynthesis of osteopontin, possibly at the level of transcription.
Animals↗
Immunohistochemical demonstration of a 44-KD phosphoprotein in developing rat bones.
Polyclonal antibodies against a 44-KD phosphoprotein (44K BPP) from rat bone were raised in rabbits, affinity-purified, and used as probes to study the protein's distribution in various types of developing bones from newborn rats. Three immunostaining procedures were applied utilizing indirect immunofluorescence, avidin-biotin-peroxidase complex, and avidin-gold complex with silver enhancement. All methods gave essentially identical and/or complementary results. Antigenicity for anti-44K BPP was detected in endochondral and membranous bone. In the latter, it was also demonstrated in the osteoid. In the woven bone of lower jaw, immunoreactivity for anti-44K BPP antibodies was found in fibroblast-shaped cells (pre-osteoblasts) that were between the bone trabeculae but not in direct contact with bony extracellular material. In addition to these presumed osteoprogenitor cells, osteoblasts as well as osteocytes were strongly stained; the cytoplasmic staining was associated with the Golgi apparatus. Occasionally immunoreactivity was detected in osteoclasts, but in these cells immunostaining was either diffusely spread in the cytoplasm or present only at sites of bone erosion. These findings support the hypothesis that the 44K BPP is a protein made by osteoblasts and is localized predominantly in bone. Furthermore, the protein appears to be expressed early in histogenesis of the bone-forming cells.
Alkaline Phosphatase↗
Resistance of the stromal cell in murine long-term bone marrow cultures to damage by ionizing radiation.
The ability of bone marrow stromal cells to survive and function after exposure to ionizing radiation remains controversial. Therefore, we used the murine long-term bone marrow culture system to analyze the effects of single doses of ionizing radiation (9-500 Gy) on the function of a preexisting, nearly confluent stroma that was supportive of hematopoiesis. Hematopoiesis ceased promptly in all the irradiated cultures and did not recover unless fresh marrow cells were inoculated. Radiation doses less than or equal to 100 Gy caused no obvious morphologic change in the cells. Total RNA, total protein, and collagen synthesis declined by 35%-60% within two days after even 9 Gy; but radiation doses up to 100 Gy caused minimal or no additional decline. Although RNA synthesis recovered nearly to normal within three weeks after radiation doses less than 100 Gy, total protein and collagen synthesis remained suppressed. Normal adherent layers irradiated with 9-50 Gy supported long-term hematopoiesis by fresh Sl/Sld marrow cells, although Sl/Sld marrow did not demonstrate sustained hematopoiesis when cultured in plain culture dishes or over normal stroma irradiated with 200 Gy. Thus, bone marrow stromal cells in long-term cultures did not show evidence of substantial cell death over at least the six-week period studied after irradiation with as much as 100 Gy, and they maintained hematopoietic supportive functions when irradiated with up to at least 50 Gy.
Animals↗
Sl/Sld mouse bone marrow stroma in vitro contains an active radiation-sensitive inhibitor of normal hemopoiesis.
Sl/Sld mice have a defective hemopoietic microenvironment. It has been assumed, based upon previous studies, that the primary abnormality in these mice is simply lack of a necessary supportive or inductive material within the hemopoietic stroma. We used in vitro long-term bone marrow cultures to characterize further the nature of the hemopoietic microenvironmental defect in Sl/Sld mice. Sl/Sld mouse bone marrow cells consistently produced less than 10% of the total hemopoietic cells and multipotent and unipotent hemopoietic progenitor cells produced in cultures of marrow from normal, congenic +/+ mice. If fresh Sl/Sld and +/+ marrow cells were mixed prior to establishing long-term marrow cultures, there was a direct correlation between number of Sl/Sld cells added and degree of inhibition of +/+ hemopoiesis. A pre-established, confluent Sl/Sld adherent stromal layer inhibited hemopoiesis by fresh +/+ marrow cells by nearly 70%, as compared with dishes with irradiated +/+ or no stroma. This inhibitory effect was abrogated by irradiation of the Sl/Sld stroma prior to addition of the fresh +/+ marrow cells. Similarly, unirradiated, but not 9 to 200 Gy irradiated Sl/Sld stroma inhibited proliferation of the factor-dependent FDC-P1 hemopoietic progenitor cell line. Thus, the Sl/Sld hemopoietic microenvironment actively inhibits hemopoiesis in vitro, and this inhibition can be at least partially eliminated by irradiation of the Sl/Sld stroma.
Anemia, Macrocytic↗
Acidic glycoproteins from bovine compact bone.
Several acidic glycoproteins with apparent molecular weights of 85,000 (bone sialoprotein, BSP), 80,000, 67,000, 60,000, 40,000 (osteonectin) and 30,000 were isolated from adult bovine compact bone. About 20-25% of the 67 K preparation was serum albumin. This contaminant was removed by passing the protein through Blue Sepharose CL-6B; the material that emerged in the void volume of this affinity column contained the bone 67 K glycoprotein free of serum albumin. All six of the proteins had high amounts of aspartic and glutamic acid. The amino acid compositions of 80 K, 67 K, 40 K and 30 K glycoproteins were very similar. The 60 K glycoprotein had an amino acid content that differed from the others with notably higher proline, alanine and valine values. All six protein samples contained mannose, galactose, glucosamine, galactosamine and sialic acid, but no fucose. The carbohydrate contents varied from about 10% for osteonectin to 38% for BSP. Antibodies were raised against electrophoretically purified osteonectin and used in enzyme-linked immunosorbent assays (ELISA). In addition to reactivity with osteonectin, the antibodies displayed strong reactivity to 80 K and 30 K glycoprotein preparations, but did not react with 67 K or 60 K bone glycoproteins or bovine serum albumin. None of the acidic glycoproteins cross-reacted with antibodies to bovine serum albumin, bovine serum proteins or human alpha 2-HS-glycoprotein.
Amino Acids↗
Proteoglycans of adult bovine compact bone.
Proteoglycans of bovine compact bone were purified by chromatography of the formic acid precipitate of an EDTA extract. The sequential chromatographic steps consisted of gel filtration on Sepharose CL-6B in 4-M guanidine HCl, ion-exchange chromatography on DEAE-Sephacel in 4-M urea and rechromatography on Sepharose CL-6B in 4-M guanidine HCl. The preparation consisted of a relatively small proteoglycan (Kav = 0.4 on Sepharose CL-6B) containing about 40% protein, 21% hexuronic acid, 23% galactosamine and lesser amounts of other monosaccharides. The core protein was shown by gradient NaDodSO4 gel electrophoresis, electrotransfer and immunodetection to be monodispersed with an Mr = 45,000. Analysis of glycopeptides obtained after papain digestion of the proteoglycan and separation from glycosaminoglycan chains by gel chromatography, indicated that both N-linked and O-linked oligosaccharides were present. The glycosaminoglycan chains liberated by papain digestion eluted from Sepharose CL-6B as a broad peak with Kav = 0.50, slightly ahead of the position of elution of bovine nasal cartilage glycosaminoglycans (Kav = 0.52); the bone glycosaminoglycans are thus slightly larger than those from cartilage and smaller than the ones attached to fetal bone proteoglycans. These chains were totally susceptible to chondroitinase AC II, a procedure that yielded unsaturated disaccharides corresponding predominantly to chondroitin-4-sulfate, and to a lesser extent chondroitin-6-sulfate. Antisera raised against adult bone proteoglycans cross-reacted with core protein of bone proteoglycan (obtained after chondroitinase digestion) but not with papain digested proteoglycan. In addition, they cross-reacted with core protein and trypsin-liberated, chondroitin sulfate rich region (AlTAl) derived from cartilage proteoglycans and, to a lesser extent, rat bone proteoglycans. No cross-reactivity could be detected to Smith-degraded cartilage proteoglycans, bone acidic glycoproteins or serum proteins.
Amino Acids↗