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Biomedical subjects

E Canalis

Publications and source records attributed to E Canalis.

At least 145 records · Page 8Linked to original sources
PubMed1988

Skeletal tissue and transforming growth factor beta.

Normal skeletal growth results from a balance between the processes of bone matrix synthesis and resorption. These activities are regulated by both systemic and local factors. Bone turnover is dynamic, and skeletal growth must be maintained throughout life. Although many growth promoters are associated with bone matrix, it is enriched particularly with transforming growth factor beta (TGF-beta) activity. Experimental evidence indicates that TGF-beta regulates replication and differentiation of mesenchymal precursor cells, chondrocytes, osteoblasts, and osteoclasts. Recent studies further suggest that TGF-beta activity in skeletal tissue may be controlled at multiple levels by other local and systemic agents. Consequently, the intricate mechanisms by which TGF-beta regulates bone formation are likely to be fundamental to understanding the processes of skeletal growth during development, maintenance of bone mass in adult life, and healing subsequent to bone fracture.

Alkaline Phosphatase↗
PubMed1988

Effects of basic fibroblast growth factor on bone formation in vitro.

Basic fibroblast growth factor (bFGF) was studied for its effects on bone formation in cultured rat calvariae. bFGF at 0.1-100 ng/ml stimulated [3H]thymidine incorporation into DNA by up to 4.4-fold. bFGF also increased the number of colcemid-induced metaphase arrested cells and the DNA content. Transient (24 h) treatment with bFGF enhanced [3H]-proline incorporation into collagen 24-48 h after the factor was removed; this effect was DNA synthesis dependent and blocked by hydroxyurea. The collagen stimulated by bFGF was type I, and this effect was observed primarily in the periosteum-free bone. In contrast, continuous treatment with bFGF for 24-96 h inhibited [3H]proline incorporation into type I collagen. bFGF did not alter collagen degradation. In conclusion, bFGF stimulates calvarial DNA synthesis, which causes an increased number of collagen-synthesizing cells, but bFGF has a direct inhibitory effect on collagen synthesis.

Animals↗
PubMed1988

Isolation and characterization of insulin-like growth factor I (somatomedin-C) from cultures of fetal rat calvariae.

Cultured bones have been shown to secrete local regulators of bone remodeling, such as beta 2-microglobulin, transforming growth factor-beta, and insulin-like growth factor (IGF), but the IGF secreted has not been characterized. In the present study, IGF from medium conditioned by 21-day-old fetal rat calvariae was isolated and characterized. IGF was purified using dialysis, gel filtration, and reverse phase HPLC. Amino acid composition was compatible with that of IGF I (somatomedin-C), and amino-terminal sequence analysis revealed homology with IGF-I. The concentration of IGF-I in the calvarial culture medium was 1 nM and was suppressed by cycloheximide. Calvaria-derived rat IGF I at 20 nM stimulated DNA and collagen synthesis by 42% and 26%, respectively, in monolayer cultures of osteoblast-rich rat parietal bone cells. This study indicates that locally produced IGF-I regulates bone formation in cultures of 21-day-old fetal rat calvariae.

Amino Acid Sequence↗
PubMed1988

Insulin-like growth factor I has independent effects on bone matrix formation and cell replication.

The effects of insulin-like growth factor-I (IGF-I) and insulin on bone matrix synthesis and bone cell replication were studied in cultured 21-day-old fetal rat calvariae. Histomorphometry techniques were developed to measure the incorporation of [2,3-3H]proline and [methyl-3H]thymidine into bone matrix and bone cell nuclei, respectively, using autoradiographs of sagittal sections of calvariae cultured with IGF-I, insulin, or vehicle for up to 96 h. To confirm an effect on bone formation, IGF-I was also studied for its effects on [3H]proline incorporation into collagenase-digestible protein (CDP) and noncollagen protein and on [3H]thymidine incorporation into acid-precipitable material (DNA). IGF-I at 10(-9)-10(-7) M significantly increased the rate of bone matrix apposition and CDP after 24 h by 45-50% and increased cell labeling by 8-fold in the osteoprogenitor cell zone, by 4-fold in the osteoblast cell zone, and by 2-fold in the periosteal fibroblast zone. Insulin at 10(-9)-10(-6) M also increased matrix apposition rate and CDP by 40-50%, but increased cell labeling by 2-fold only at a concentration of 10(-7) M or higher and then only in the osteoprogenitor cell zone. When hydroxyurea was added to IGF-I-treated bones, the effects of IGF-I on DNA synthesis were abolished, but the increase in bone matrix apposition induced by IGF-I was only partly diminished. In conclusion, IGF-I stimulates matrix synthesis in calvariae, an effect that is partly, although not completely, dependent on its stimulatory effect on DNA synthesis.

Animals↗
PubMed1988

Tumor necrosis factor-alpha inhibits collagen synthesis and alkaline phosphatase activity independently of its effect on deoxyribonucleic acid synthesis in osteoblast-enriched bone cell cultures.

Tumor necrosis factor-alpha (TNF alpha), a product of activated monocytes, induces tissue wasting in certain solid tumors in vivo and in in vitro model systems. Recent studies indicate that TNF alpha also regulates cell replication and expression of differentiated function in a variety of nonneoplastic cell systems. Since monocyte products could accumulate in bone with trauma, inflammation, or other disease states, bone cell activity might be altered by the presence of these pathophysiological molecules. Using cells obtained by sequential enzyme release from fetal rat parietal bone, we find that TNF alpha has acute stimulatory and inhibitory effects on bone cell macromolecular synthesis. Within 24 h of exposure, recombinant human TNF alpha at 0.3-100 nM progressively increases the rate of DNA synthesis in osteoblast-enriched cell cultures up to 3- to 4-fold, and 3-100 nM TNF alpha reduces collagen production and alkaline phosphatase activity by 20-30%. These decreases are not altered by 1 mM hydroxyurea, which blocks the mitogenic effect of TNF alpha by 85-90%. In addition, hydroxyproline levels in the culture medium do not increase relative to the control value after TNF alpha treatment, suggesting that decreased collagen production results from less synthesis rather than increased collagen degradation. Hybridization studies with cDNA encoding the alpha 1-chain of rat type I collagen show that TNF alpha increases type I collagen mRNA to an extent similar to its effect on cell replication. Therefore, TNF alpha appears to inhibit collagen synthesis and alkaline phosphatase activity in osteoblast-enriched cell cultures by mechanisms that are not related to its effects on cell replication.

Alkaline Phosphatase↗
PubMed1987

Transforming growth factor beta is a bifunctional regulator of replication and collagen synthesis in osteoblast-enriched cell cultures from fetal rat bone.

Transforming growth factor beta (TGF beta) stimulates cell replication in fetal rat calvariae, and studies with isolated bone cells suggest that the primary mitogenically responsive cell is of the osteoblast lineage. The effect of TGF beta on bone cell replication is biphasic and depends on both the TGF beta concentration and cell density in monolayer culture. After 23 h of treatment, DNA synthesis in confluent cells is progressively enhanced by 0.15-15 ng/ml TGF beta; but in subconfluent cells, 15 ng/ml is less than maximal; and in sparse cell cultures, it is inhibitory. At all cell densities, however, 15 ng/ml TGF beta stimulates collagen synthesis, an effect which is more pronounced when DNA synthesis rates are declining. Furthermore, 1 mM hydroxyurea, which blocks the mitogenic effect of TGF beta by 85%, only minimally influences the increase in collagen synthesis. Cytoplasmic slot blot analysis reveals alterations in the amount of type I collagen mRNA in TGF beta-treated cells, suggesting that control is exerted, at least in part, at the transcriptional level. Since TGF beta is found in culture medium conditioned by bone explants and in bone tissue extracts, these results support that TGF beta is an important and multifunctional autocrine regulator of bone formation.

Animals↗
PubMed1987

Isolation of EGF-dependent transforming growth factor (TGF beta-like) activity from culture medium conditioned by fetal rat calvariae.

A transforming growth factor of the beta class (TGF-beta), defined by its ability to induce normal rat kidney cells (NRK, clone 49F) to form anchorage-independent large colonies in soft agar in the obligate presence of epidermal growth factor, has been prepared from culture medium conditioned by fetal rat calvariae. This activity was purified by acetic acid extraction, gel permeation chromatography, and two reversed-phase HPLC (rpHPLC) steps. Bone culture derived-TGF beta-like activity was soluble in 1.0 M acetic acid, eluted from Sephadex G-75 at relative molecular mass (Mr) 25,000, from mu Bondapak C18 rpHPLC at 63 +/- 5% methanol in 0.1 M acetic acid, and from mu Bondapak CN rpHPLC at 36 +/- 2% n-propanol in 0.1% trifluoroacetic acid. Based on specific activity estimations at each stage of purification, TGF beta-like activity was purified 2500-fold with a 14% recovery, and 1 l of conditioned medium yielded 1-2 micrograms of factor. Silver-stained polyacrylamide gels of this material after CN mu Bondapak rpHPLC revealed a predominant band of (Mr) 24,000.

Animals↗
PubMed1987

Mitogenesis in fetal rat bone cells simultaneously exposed to type beta transforming growth factor and other growth regulators.

Type beta transforming growth factor (TGF-beta) is found in large amounts in bone tissue, and is a potent mitogen for osteoblast-enriched cell cultures obtained from fetal rat parietal bone. Because other local and systemic factors may be presented to bone cells simultaneously with TGF-beta, it is important to understand the effects of this complex growth regulator in such circumstances. Unlike the effects observed in many tissue systems, TGF-beta does not invariably inhibit the mitogenic response of bone cells to other growth promoters. In contrast, other factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and type alpha tumor necrosis factor (TNF-alpha) limit the response of osteoblastic bone cells to TGF-beta. TGF-beta is a much weaker mitogen for fibroblastic cells obtained from fetal rat bone, whereas fetal bovine serum, EGF, bFGF, and TNF-alpha are more potent stimulators. In addition, TGF-beta does not significantly impair the response of the fibroblastic bone cells to the other tested agents. These findings reinforce a role of TGF-beta as an anabolic bone growth regulator, and suggest that its function may be modified by other local or systemic agents that can also affect bone cells.

Animals↗
PubMed1987

Effects of endothelial cell growth factor on bone remodelling in vitro.

Endothelial cell growth factor (ECGF) alpha was studied for its effects on bone formation in cultured fetal rat calvariae and on bone resorption in cultured fetal rat long bones. ECGF at 0.1-100 ng/ml stimulated [3H]thymidine incorporation into DNA, an effect enhanced by heparin. Treatment with ECGF for 24 h decreased the incorporation of [3H]proline into collagen but treatment for 48-96 h increased collagen and noncollagen protein synthesis, an effect that was concomitant with an increase in DNA content. ECGF did not alter collagen degradation in calvariae or 45Ca release from long bones, which indicated it had no effect on bone resorption. Although ECGF increased prostaglandin E2 concentrations, its effect on DNA synthesis was not prostaglandin-mediated. In conclusion, ECGF stimulates calvarial DNA synthesis, which is an effect that results in a generalized increase in protein synthesis, but ECGF has no effect on matrix degradation or bone resorption.

Animals↗
PubMed1987

A bone-derived growth factor isolated from rat calvariae is beta 2 microglobulin.

Rat calvariae are known to secrete a bone-derived growth factor (BDGF) that stimulates bone DNA and collagen synthesis. BDGF was purified from calvarial culture medium with dialysis, gel filtration and HPLC. Amino acid composition of BDGF was compatible with murine beta 2 microglobulin (beta 2 m), and amino terminal sequence analysis revealed identity with mature murine beta 2 m. This was confirmed by Western blot analysis using a polyclonal antibody to beta 2 m. Like BDGF, human beta 2 m stimulated bone DNA, collagen and noncollagen protein synthesis. Thus, BDGF, an autologous regulator of bone formation, is homologous to beta 2 m.

Amino Acid Sequence↗
PubMed1987

Effects of tumor necrosis factor on bone formation in vitro.

Tumor necrosis factor (TNF) was studied for its effects on bone formation in cultured rat calvariae. TNF alpha at 100-100,000 U/ml stimulated [3H]thymidine incorporation into DNA, an effect that appeared after 24 h of treatment and lasted 96 h. Transient (24-h) treatment with TNF alpha increased [3H]proline incorporation into type I collagen 24-72 h after the factor was removed; this effect was DNA synthesis dependent and blocked by hydroxyurea. Transient treatment with TNF alpha also increased alkaline phosphatase activity. In contrast, continuous treatment with TNF alpha for 48-96 h caused a marked inhibition on [3H]proline incorporation into type I collagen and alkaline phosphatase activity. TNF alpha caused a small increase in collagen degradation. Lymphotoxin had similar effects to those of TNF alpha. In conclusion, TNF alpha stimulates calvarial DNA synthesis which causes an increased number of collagen-synthesizing cells, but TNF alpha has a direct inhibitory effect on osteoblastic function.

Alkaline Phosphatase↗
PubMed1986

Interleukin-1 has independent effects on deoxyribonucleic acid and collagen synthesis in cultures of rat calvariae.

Interleukin-1 (IL-1), a monokine known to be important in host defense mechanisms and recently reported to stimulate bone resorption, was studied for its effects on bone formation in cultures of 21-day-old fetal rat calvariae. IL-1 at 0.1-5 U/ml stimulated the incorporation of [3H] thymidine into acid-insoluble residues (DNA) by 29-123% in calvariae treated for 24-96 h. IL-1 also increased the bone DNA content and the number of mitoses after colcemid arrest. IL-1 stimulated total protein synthesis. Treatment with IL-1 at 0.01-1 U/ml for 24 h caused a small increase in the incorporation of [3H]proline into collagenase-digestible protein (CDP) and non-collagen protein (NCP). However, higher doses of IL-1 (5 U/ml) or longer exposure to the agent (1 U/ml for 96 h) inhibited the labeling of CDP but not of NCP. IL-1 affected only type I collagen. The stimulatory effects of IL-1 on DNA, CDP, and NCP labeling were independent, since they were observed at different doses, and hydroxyurea abolished the effect on DNA without changing that on CDP and NCP labeling. Indomethacin blocked the stimulatory effect on CDP and NCP labeling, suggesting a prostaglandin-mediated effect, but did not change the IL-1 effect on DNA synthesis. These studies indicate that IL-1 stimulates calvarial DNA, collagen, and NCP synthesis, but exposure of the calvariae to high IL-1 doses or to IL-1 for prolonged periods of time results in an inhibition of collagen synthesis.

Animals↗
PubMed1986

Isolation of a nontransforming bone-derived growth factor from medium conditioned by fetal rat calvariae.

Previous studies have indicated that medium conditioned by 21-day-old fetal rat calvariae contains bioactive proteins termed bone-derived growth factors (BDGF) I and II. In the present studies we have purified the nontransforming BDGF II by dialysis, molecular sieving, three reverse phase HPLC steps, and preparative polyacrylamide gel electrophoresis. The second HPLC step (HPLC-2) yielded a recovery of 22% of the biological activity and achieved a 1500-fold purification, resulting in 20 micrograms protein/liter calvarial conditioned medium; the third HPLC step was of limited value in the purification of BDGF. Analytical PAGE revealed that the majority of the protein in HPLC-2-purified BDGF migrated with a relative molecular mass (Mr) of 11,000 and two additional proteins were seen at a Mr of 22,000-23,000. On preparative PAGE, the material migrating with a Mr of 11,000 stimulated parameters of bone and fibroblast growth in vitro, whereas the material with a Mr of 22,000-23,000 had less biological activity. Isoelectric focusing revealed that BDGF had an isoelectric point (pI) of 5. BDGF enhanced the incorporation of [3H]thymidine into DNA in fibroblast and calvarial cultures and of [3H]proline into collagen and noncollagen protein in calvariae. In conclusion, fetal rat calvariae secrete a BDGF with an estimated Mr of 11,000 and a pI of 5; this material stimulates bone and fibroblast growth in vitro.

Animals↗
PubMed1986

Human platelet-derived transforming growth factor-beta stimulates parameters of bone growth in fetal rat calvariae.

Human platelet-derived transforming growth factor type beta (TGF beta) is mitogenic for fetal rat calvariae in serum-free organ culture. It enhances DNA synthesis in short (24-h) and long (48- to 96-h) term cultures, but produces no significant stimulatory effects on bone collagen synthesis or alkaline phosphatase activity (two parameters of differentiated osteoblastic cell-type function) when present continuously in culture. Transitory treatment with TGF beta, however, induces a subsequent stimulation of collagen and noncollagen protein synthesis that depends on prior cell replication, suggesting an increase in the number of newly differentiated bone cells. In addition, TGF beta increases prostaglandin release, but this effect is probably unrelated to its mitogenic function. TGF beta activity is also found in culture medium conditioned by fetal rat calvariae, and the bone-derived factor produces effects similar to those of the human platelet factor. This polypeptide, therefore, may have an important function in early stages of bone development as well as bone repair after trauma-induced platelet degranulation.

Animals↗
PubMed1985

Studies of hormonal regulation of osteocalcin synthesis in cultured fetal rat calvariae.

The synthesis of osteocalcin, the major non-collagenous protein of adult bone, was examined in cultures of 21-day fetal rat calvariae. Osteocalcin was measured by a sensitive and specific radioimmunoassay. Osteocalcin concentration in unincubated calvariae was 14.5 +/- 0.5 ng/calvaria. After incubation, there was a continuous increase in bone and medium osteocalcin, and by 96 h the values were about 100% higher than in unincubated calvariae. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) at 10(-11) to 10(-8)M increased osteocalcin synthesis. The effect appeared as early as 6 h after treatment and was primarily observed in the culture medium, and 1,25-(OH)2D3 stimulated osteocalcin up to 9-fold by 96 h. Concomitant with the effect on osteocalcin synthesis, 1,25-(OH)2D3 inhibited collagen synthesis. Cycloheximide markedly decreased osteocalcin concentrations in control and 1,25-(OH)2D3-treated calvariae. The stimulatory effect on osteocalcin synthesis was specific to 1,25-(OH)2D3 since 24,25-dihydroxyvitamin D3, parathyroid hormone, epidermal growth factor, and prostaglandin E2 did not stimulate osteocalcin synthesis, and parathyroid hormone and epidermal growth factor opposed the 1,25-(OH)2D3 stimulatory effect. Insulin did not alter osteocalcin concentration by itself but enhanced the effect of 1,25-(OH)2D3. In conclusion, 1,25-(OH)2D3 stimulates osteocalcin synthesis in cultures of normal calvariae, but this effect is not shared by other hormones known to affect bone metabolism.

Animals↗
PubMed1985

1,25-Dihydroxyvitamin D3 effects on collagen and DNA synthesis in periosteum and periosteum-free calvaria.

1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is essential for normal growth and mineralization, but its direct effects on various aspects of bone formation remain controversial. 1,25(OH)2D3 was studied for its effects on DNA, collagen and noncollagen protein synthesis, and alkaline phosphatase activity (APA) in the periosteum and periosteum-free bone from 21-day fetal rat calvariae. 1,25(OH)2D3 (0.01 to 10 nM) inhibited the incorporation of 3H-proline into collagenase-digestible protein (CDP) and the percent of collagen synthesized, and, at 10 nM, APA in the periosteum-free bone. 1,25(OH)2D3 inhibited type I collagen without affecting other collagen types. In contrast, 1,25(OH)2D3 at 10 nM caused a small but significant stimulation of the incorporation of 3H-thymidine into acid-insoluble residues (DNA) and on DNA content; both effects were exclusively observed in the periosteum. Hydroxyurea did not modify the inhibitory effect of 1,25(OH)2D3 on 3H-proline incorporation into CDP. These studies indicate that 1,25(OH)2D3 stimulates periosteal DNA synthesis but inhibits type I collagen synthesis and APA in the periosteum-free bone.

Alkaline Phosphatase↗
PubMed1985

Transforming and nontransforming growth factors are present in medium conditioned by fetal rat calvariae.

Conditioned medium recovered from fetal rat calvarial cultures contains an autocrine factor termed bone-derived growth factor (BDGF); this factor has been purified by acid extraction, gel-permeation chromatography, and two reversed-phase HPLC steps and examined for mitogenicity on normal rat kidney fibroblasts (NRK, clone 49F). HPLC-purified BDGF caused a dose-related increase in cell number, DNA content, and [3H]thymidine incorporation into acid-insoluble material. Since highly purified BDGF appeared less mitogenic than cruder preparations, the latter were tested for additional growth factors, with particular attention to those required for anchorage-independent colony formation in soft agar. BDGF did not displace 125I-labeled epidermal growth factor (EGF) in a radioligand-receptor assay, indicating the absence of EGF and transforming growth factor alpha (TGF-alpha). Without EGF, no BDGF preparation induced NRK cells to form soft agar colonies. However, calvarial conditioned medium contained a factor which, like TGF-beta, induced large soft-agar colonies in the presence of EGF; this TGF-beta-like factor did not copurify with BDGF. Polyclonal antibodies against platelet-derived growth factor did not neutralize the effects of BDGF on NRK cells. BDGF is a potent mitogen for nonskeletal-tissue-derived fibroblasts. Although crude BDGF preparations do contain TGF-beta, BDGF is distinct from this factor and others necessary for NRK cell transformation to anchorage-independent growth.

Animals↗