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L F Bonewald

Publications and source records attributed to L F Bonewald.

At least 55 records · Page 3Linked to original sources
PubMed1995

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.

Animals↗
PubMed1994

Latent transforming growth factor-beta is produced by chondrocytes and activated by extracellular matrix vesicles upon exposure to 1,25-(OH)2D3.

Resting zone and growth zone (GC) costochondral chondrocytes constitutively release latent, but not active, transforming growth factor-beta (TGF-beta) into the culture medium. When exogenous TGF-beta is added to the culture medium, no autocrine effect is observed. However, when 1,25-(OH)2D3 is added, a dose-dependent inhibition of latent TGF-beta release is found. Messenger RNA levels for TGF-beta 1 are unchanged by treatment with either 1,25-(OH)2D3 or TGF-beta 1. Since active growth factor was not observed in the conditioned medium, we tested the hypothesis that latent TGF-beta could be activated in the matrix. GC matrix vesicles, extracellular organelles associated with matrix calcification, were able to activate latent TGF-beta 1 and TGF-beta 2 when preincubated with 1,25-(OH)2D3. In contrast, GC plasma membranes activated latent TGF-beta, and addition of 1,25-(OH)2D3 inhibited this activation. The 1,25-(OH)2D3-dependent decrease in latent TGF-beta in the medium, with no detectable change in mRNA level, and the inhibition of plasma membrane activation of latent TGF-beta by 1,25-(OH)2D3 suggest that 1,25-(OH)2D3 may act through post-transcriptional and/or nongenomic mechanisms. The results also suggest that latent TGF-beta is activated in the matrix and that 1,25-(OH)2D3 regulates this activation by a direct, nongenomic action on the matrix vesicle membrane.

24,25-Dihydroxyvitamin D 3↗
PubMed1994

Characterization and autoregulation of latent transforming growth factor beta (TGF beta) complexes in osteoblast-like cell lines. Production of a latent complex lacking the latent TGF beta-binding protein.

We have previously shown that bone organ cultures produce large amounts of latent transforming growth factor beta (TGF beta), which lacks latent TGF beta-binding protein (LTBP). In this study we used the known osteoblast-like cell lines UMR-106, ROS 17/2.8, and MG63 as models to further examine latent TGF beta expression in bone. We found that the osteosarcoma cell line UMR-106 secreted latent TGF beta almost exclusively as a 100-kDa complex lacking LTBP. ROS 17/2.8 cells produced both the 100-kDa complex and also a 290-kDa complex containing the fibroblastic (190 kDa) form of LTBP. MG63 cells (like human foreskin fibroblasts) expressed almost exclusively the 290-kDa complex. To investigate the regulation of latent TGF beta complexes in bone cells we assessed the effects of TGF beta 1 treatment on expression of active and latent TGF beta. TGF beta 1 induced secretion of latent but not active TGF beta in all cell types examined. In human foreskin fibroblast cells, TGF beta 1 and LTBP mRNA were expressed concomitantly. In contrast, in osteosarcoma cell lines autoinduction of TGF beta 1 mRNA was associated with either a delayed increase or no change in LTBP mRNA. In UMR-106 cells LTBP message was virtually undetectable. We postulate that the expression of different latent TGF beta forms by osteoblast-like cells may reflect their maturation states and that different latent TGF beta complexes may have different functions, for example as secretory forms or as matrix storage forms.

Animals↗
PubMed1994

Effects of transforming growth factor beta on bone nodule formation and expression of bone morphogenetic protein 2, osteocalcin, osteopontin, alkaline phosphatase, and type I collagen mRNA in long-term cultures of fetal rat calvarial osteoblasts.

Transforming growth factor beta (TGF-beta) is one of the most abundant of the known growth regulatory factors stored within the bone matrix. When bone is resorbed, TGF-beta is released in an active form and is a powerful bone growth stimulant. When injected into the subcutaneous tissue over the calvarial surface of rodents, it rapidly causes proliferation of the periosteal layer and accumulation of new woven bone. In this report, we describe the effects of TGF-beta 1 on first subcultures of fetal rat osteoblasts obtained from calvarial bones and cultured from confluence with ascorbic acid and beta-glycerophosphate. Under these conditions, nodules with characteristics of normal bone appear by day 8. Similar to experiments described by Antosz et al., TGF-beta added to confluent cultures inhibited the formation of bone nodules. Both the number and total area of the nodules were quantitated and shown to be completely inhibited by 2 ng/ml of TGF-beta 1. TGF-beta also impaired the expression of genes associated with bone formation, including type I collagen, alkaline phosphatase, osteopontin, and osteocalcin. TGF-beta also inhibited the expression of mRNA for the bone morphogenetic protein 2 (BMP-2). These results, showing suppression of markers representative of osteoblast differentiation, suggest that the effects of TGF-beta to stimulate bone formation in vivo are not likely a result of effects on differentiated mineralizing osteoblasts but, as suggested by previous studies, more likely are caused by effects on osteoblast precursors. These results also suggest that endogenous BMP-2 expression in fetal rat calvaria cells is important for bone cell differentiation.

Alkaline Phosphatase↗
PubMed1994

Role of active and latent transforming growth factor beta in bone formation.

At first reading the statement "TGF beta stimulates bone formation but inhibits mineralization" may appear to be an oxymoron. However, the bone formation process can take weeks to months to complete, and the unique properties of TGF beta allow this factor to be stored in bone matrix in a latent form, ready to be activated and inactivated at key, pivotal stages in this long process. TGF beta may act to trigger the cascade of events that ultimately leads to new bone formation. However, once this process is initiated, TGF beta must then be inactivated or removed because if present in the later stages of bone formation, mineralization will be inhibited. The unique properties of TGF beta and its role in bone remodeling are the subject of this review.

Animals↗
PubMed1993

5-Lipoxygenase metabolites of arachidonic acid stimulate isolated osteoclasts to resorb calcified matrices.

Bone resorption requires cooperation between osteoclasts and mononuclear accessory cells by mechanisms which have not been elucidated. Since multinucleated cells in giant cell tumors of bone have many phenotypic and functional characteristics of normal osteoclasts, we have examined the interaction between the bone-resorbing multinucleated cells and the distinct mononuclear stromal cells from these tumors. We have found that these mononuclear cells produce an activity which stimulates both giant cells from giant cell tumors and rodent osteoclasts to resorb bone in vitro. We have identified the activity and found that it represents several products of the 5-lipoxygenase pathway of arachidonic acid metabolism, namely 5-hydroxyeicosatetraenoic acid and the leukotrienes. These data indicate that 5-lipoxygenase metabolites stimulate isolated osteoclasts to resorb bone in vitro and may represent a mechanism by which mononuclear stromal cells in human giant cell tumors communicate with the giant cells. In addition, these results may explain a possible mechanism for communication between accessory cells and osteoclasts involved in normal bone resorption.

Acid Phosphatase↗
PubMed1993

Interleukin-1 receptor antagonist inhibits the hypercalcemia mediated by interleukin-1.

Recently, the effects of interleukin-1 (IL-1) on bone resorption in organ culture have been shown to be inhibited by an interleukin-1 receptor antagonist (IL-1RA), a novel monocyte cytokine in the IL-1 family. IL-1RA, which binds to IL-1 receptors and inhibits many of the effects of IL-1 alpha and beta, has been purified, cloned, and expressed. We used IL-1RA to investigate its effects on calcium homeostasis in vivo. After confirming that IL-1RA completely inhibited the effects of IL-1 on bone resorption in organ cultures, we tested the effects of IL-1RA on hypercalcemia mediated by IL-1 in normal mice and found that prolonged hypercalcemia provoked by IL-1 was completely inhibited by IL-1RA. The initial transient decrease in blood ionized calcium observed following an injection of IL-1 was also abrogated. IL-1RA had no effect alone on blood ionized calcium or on hypercalcemia mediated by parathyroid hormone (PTH) or PTH-related protein (PTHrP). These data suggest that antagonists to the IL-1 receptor may provide a useful therapeutic approach to osteoclastic bone resorption and hypercalcemia that is IL-1 dependent.

Animals↗
PubMed1993

Evidence that interleukin-1 mediates its effects on bone resorption via the 80 kilodalton interleukin-1 receptor.

Interleukin-1 (IL-1) mediates its effects through two distinct receptors, one of 80 kilodaltons (80 kD) present in athymic lymphocytes and fibroblasts, and one of 60 kD present in cells of the monocyte-macrophage lineage. A novel monocyte cytokine in the IL-1 family which binds to both the 80 and the 60 kD receptors has been purified, cloned, and expressed. As the interleukin-1 receptor antagonist (IL-1ra) has been shown to inhibit bone resorption in organ culture, it is not clear whether these effects are mediated through the 80 or the 60 kD receptor. Recently, neutralizing antibodies (35F5) have been developed to the 80 kD receptor which inhibit IL-1 effects mediated through this receptor. To determine the importance of the 80 kD receptor to IL-1-mediated bone resorption, we used the neutralizing antibodies (35F5) to the 80 kD receptor to determine if they inhibited bone resorption stimulated by IL-1 in bone organ cultures. The 35F5 antibody blocked bone-resorbing activity due to IL-1 completely, and also blocked control or "endogenous" bone-resorbing activity present in murine bone organ cultures incubated in control media. The 35F5 antibody had no effect on bone resorption mediated by tumor necrosis factor (TNF), or parathyroid hormone (PTH). These data suggest that the availability of the 80 kD IL-1 receptor is required for osteoclastic bone resorption mediated by IL-1.

Animals↗
PubMed1993

Direct effects of transforming growth factor-beta on chondrocytes are modulated by vitamin D metabolites in a cell maturation-specific manner.

Chondrocytes in the endochondral differentiation pathway produce transforming growth factor-beta (TGF-beta) and response to this growth factor both in vitro and in vivo. To clarify the role that cell maturation state plays in the response, we used a well characterized chondrocyte cell culture model which compares cartilage cells at two different stages of maturation. Confluent fourth passage cultures of rat costochondral resting zone and growth zone cartilage cells were incubated with recombinant human (rh) TGF-beta-1 for 24, 48, or 72 h, and the effect on cell number and [3H]thymidine incorporation was observed. To assess whether TGF-beta regulates chondrocyte differentiation to a calcifying cartilage phenotype, cells were incubated for 24 h with TGF-beta, and the specific activities of alkaline phosphatase and phospholipase A2, two enzymes associated with calcification, were assayed in isolated plasma membranes and matrix vesicles. Alkaline phosphatase-specific activity was also measured in the cell layer. Modulation of TGF-beta action by vitamin D metabolites, also known to regulate endochondral differentiation, was examined. The ability of the chondrocytes to produce latent TGF-beta was assayed. The results show that: 1) quiescent chondrocytes at two stages of endochondral maturation respond to rhTGF-beta-1 by increasing [3H]thymidine incorporation; 2) growth zone cells exhibit no increase in cell number over a 72-h incubation with TGF-beta, whereas resting zone cells exhibit a dose-dependent increase in cell number at 72 h; 3) nonquiescent cells exhibit an increase in alkaline phosphatase-specific activity at 24 h; 4) the effects on this membrane enzyme are comparable in the plasma membranes and matrix vesicles, but the net effect is greater in the extracellular organelle due to the intrinsically higher levels of activity; 5) although differentiation is promoted in resting zone cells, it is limited in the growth zone cells by inhibition of phospholipase A2 activity; and 6) there is a synergistic enhancement of resting zone chondrocyte differentiation when cells are exposed to rhTGF-beta-1 and 24,25-dihydroxyvitamin D3.

24,25-Dihydroxyvitamin D 3↗
PubMed1993

Characterization of a cell line derived from a human giant cell tumor that stimulates osteoclastic bone resorption.

Giant cell tumors of bone are common but unusual tumors that are comprised of multiple cell types. Most attention has been focused on the giant cells, which resemble osteoclasts morphologically and functionally. This study examines the properties of a cell line derived from mononuclear cells from one of these tumors, since it appears likely that these cells may be able to influence the activities of cells with the osteoclast phenotype. This cell line, C433, has the following characteristics: (1) it represents undifferentiated cells, not recognized by any known antigenic markers for leukocytes; (2) it contains tartrate-resistant acid phosphatase; (3) it responds to the osteotropic factors 1,25 dihydroxyvitamin D3, insulin-like growth factor I and II, but not to parathyroid hormone; (4) it forms sarcomas in nude mice; and (5) it produces an activity that stimulates isolated avian and rat osteoclasts to resorb bone. This cell line may be useful in examining interactions between osteoclasts and accessory cells involved in bone resorption.

Alkaline Phosphatase↗
PubMed1992

Effects of combining transforming growth factor beta and 1,25-dihydroxyvitamin D3 on differentiation of a human osteosarcoma (MG-63).

Transforming growth factor beta (TGF beta) and 1,25-dihydroxyvitamin D3 (1,25D3), when added simultaneously to a human osteosarcoma cell line, MG-63, induce alkaline phosphatase activity 40-70-fold over basal levels, 6-7-fold over 1,25D3 treatment alone, and 15-20-fold over TGF beta treatment alone. TGF beta and 1,25D3 synergistically increased alkaline phosphatase specific activity in both matrix vesicles and plasma membrane isolated from the cultures, but the specific activity was greater in and targeted to the matrix vesicle fraction. Inhibitor and cleavage studies proved that the enzymatic activity was liver/bone/kidney alkaline phosphatase. Preincubation of MG-63 cells with TGF beta for 30 min before addition of 1,25D3 was sufficient for maximal induction of enzyme activity. Messenger RNA for liver/bone/kidney alkaline phosphatase was increased 2.1-fold with TGF beta, 1.7-fold with 1,25D3, and 4.8-fold with the combination at 72 h. Human alkaline phosphatase protein as detected by radioimmunoassay was stimulated only 6.3-fold over control levels with the combination. This combination of factors was tested for their effect on production of three other osteoblast cell proteins: collagen type I, osteocalcin, and fibronectin. TGF beta inhibited 1,25D3-induced osteocalcin production, whereas both factors were additive for fibronectin and collagen type I production. TGF beta appears to modulate the differentiation effects of 1,25D3 on this human osteoblast-like cell and thereby retain the cell in a non-fully differentiated state.

Alkaline Phosphatase↗
PubMed1992

Expression of human transforming growth factor alpha by Chinese hamster ovarian tumors in nude mice causes hypercalcemia and increased osteoclastic bone resorption.

Transforming growth factor alpha (TGF-alpha) is a polypeptide regulator of cell growth produced by many malignant tumors. It stimulates osteoclastic resorption in bone organ culture and osteoclast-like cell formation in marrow culture. To determine whether tumor production of TGF-alpha can cause hypercalcemia in vivo, we used Chinese hamster ovarian (CHO) cells transfected with the human TGF-alpha gene (TCHO), which stably express and secrete TGF-alpha. We used nontransfected CHO cells as controls (CCHO). TCHO and CCHO were inoculated intramuscularly into one hindlimb of nude mice and grew as local solid tumors. After 4 weeks of TCHO tumor growth, plasma ionized calcium (Ca2+) increased to reach 1.48 +/- 0.03 mM (mean +/- SEM), whereas mice bearing similarly sized CCHO tumors and non-tumor-bearing mice (NTB) remained normocalcemic (normal range for Ca2+, 1.15-1.30 mM). Plasma TGF-alpha was undetectable by an ELIFA assay in all NTB mice, was markedly increased in all TCHO mice (5.75 +/- 0.78 ng/ml), and was slightly increased in CCHO mice (0.50 +/- 0.22 ng/ml). Quantitative bone histomorphometry showed a prominent increase in osteoclastic bone resorption in TCHO mice. These data suggest that TGF-alpha is a mediator of hypercalcemia and increased osteoclastic bone resorption in tumors that produce it in sufficient quantity.

Animals↗
PubMed1992

Stimulation of matrix vesicle enzyme activity in osteoblast-like cells by 1,25(OH)2D3 and transforming growth factor beta (TGF beta).

After demonstrating the presence of matrix vesicles in three osteosarcoma cell lines, MG-63, ROS 17/2.8 and MC-3T3-E1, we sought to determine whether two major enzymes localized to matrix vesicles, alkaline phosphatase and phospholipase A2, could be regulated by 1,25(OH)2D3 and/or TGF beta. Intravesicular calcification is probably dependent on these two enzymes. Alkaline phosphatase is essential for hydrolysis of phosphate-containing substrates and phospholipase A2 hydrolyzes diacylphosphatides in a calcium-mediated manner at lipid-aqueous interfaces leading to changes in membrane fluidity and possibly breakdown of the matrix vesicle. The 1,25(OH)2D3 induced increase of alkaline phosphatase in bone cells is localized to the matrix vesicle. TGF beta also increased alkaline phosphatase activity in two of the cell lines, MG-63 and ROS 17/2.8 but to a greater degree than 1,25(OH)2D3. Matrix vesicle alkaline phosphatase activity exhibited a greater response than that in the plasma membrane. TGF beta increased phospholipase A2 activity in both matrix vesicles and plasma membranes, therefore, no targeting was observed with respect to this enzyme. When TGF beta was combined with 1,25(OH)2D3, 1,25(OH)2D3 had no effect on phospholipase A2 and did not interfere with TGF beta stimulation of phospholipase A2 activity. When 1,25(OH)2D3 and TGF beta were combined, a tremendous synergy was observed in alkaline phosphatase specific activity in both plasma membranes and matrix vesicles with targeting to matrix vesicles. Therefore, TGF beta not only plays an important role in matrix formation and differentiation, but works in conjunction with 1,25(OH)2D3 to greatly potentiate the effects seen with 1,25(OH)2D3 alone.

Alkaline Phosphatase↗
PubMed1992

Chlamydia trachomatis pneumonia induces in vivo production of interleukin-1 and -6.

Cytokine induction during Chlamydia trachomatis pneumonia may alter the pathogenesis or course of disease. We examined interleukin-1 (IL-1) and IL-6 production by measuring mRNA and bioactivity in murine lungs. mRNA and bioactivity for IL-1 alpha, IL-1 beta, and IL-6 increased after Chlamydia infection. These cytokines may be important in regulating host defenses against C. trachomatis.

Animals↗