Search PubMed⌕ Search

Biomedical subjects

E Canalis

Publications and source records attributed to E Canalis.

At least 127 records · Page 7Linked to original sources
PubMed1990

Differential effects of continuous and transient treatment with parathyroid hormone related peptide (PTHrp) on bone collagen synthesis.

Parathyroid hormone-related peptide (PTHrp), a polypeptide synthesized by tumors associated with hypercalcemia and known to cause bone resorption, was examined for its effects on bone formation in cultures of 21-day fetal rat calvariae. Continuous treatment with PTHrp for 24-72 h stimulated DNA synthesis, but inhibited [3H] proline incorporation into collagen by about 50%. In contrast, transient exposure to PTHrp at 0.1-1.0 nM for 24 h followed by removal of the factor for 48 h caused an increase in [3H]proline incorporation into collagen and noncollagen protein by 2- and 1.6-fold, respectively. The stimulatory effect was seen in the periosteum-free bone, and was decreased, but not prevented by hydroxyurea. PTHrp at 1-10 nM for 24 h increased medium insulin-like growth factor (IGF) I levels by 2.5-4.4-fold, and the effect was sustained 48 h after the removal of the agent. An IGF I neutralizing antibody prevented the stimulatory effect of PTHrp on bone collagen synthesis. PTH had the same stimulatory effects as those of PTHrp on bone collagen synthesis and IGF I concentrations, although slightly lower doses were needed to observe the enhancement of [3H]proline incorporation into collagen. It is concluded that continuous treatment with PTHrp inhibits, whereas transient treatment stimulates, collagen synthesis; the stimulatory effect appears mediated by an enhancement in the local production of IGF I.

Animals↗
PubMed1989

Beta 2-microglobulin enhances insulin-like growth factor I receptor levels and synthesis in bone cell cultures.

Beta 2-Microglobulin (beta 2m), a component of the major histocompatibility complex in chordates, has growth factor-like activity for cultured rat cells. We presently describe interactions between beta 2m and insulin-like growth factor I (IGF I) in osteoblast-enriched cultures. beta 2m increased DNA synthesis and was synergistic with IGF I. Affinity labeling revealed that beta 2m enhanced IGF I receptor number, and Northern analysis and radioimmunoassay showed that beta 2m increased steady state IGF I transcripts and medium IGF I polypeptide levels. These results indicate that the growth-promoting activity of beta 2m is mediated at least in part by regulating local IGF I binding and synthesis by skeletal cells.

Animals↗
PubMed1989

Effects of platelet-derived growth factor on bone formation in vitro.

Platelet-derived growth factor (PDGF) is a polypeptide found in a variety of tissues, including bone, where it could act as an autologous regulator of skeletal remodeling. Therefore, a recombinant B chain homodimer of human PDGF was studied for its effects on bone formation in cultured rat calvariae. PDGF at 10-100 ng/ml stimulated [3H]thymidine incorporation into DNA by up to sixfold and increased the DNA content and the number of colcemid-induced metaphase arrested cells. This effect was observed in the fibroblast and precursor cell-rich periosteum. As a result of its mitogenic actions, PDGF enhanced [3H]proline incorporation into collagen, an effect that was observed primarily in the osteoblast-rich central bone. The effect of PDGF was not specific for collagen since it also increased noncollagen protein synthesis. In addition, PDGF increased bone collagen degradation. PDGF and insulin-like growth factor (IGF) I had additive effects on calvarial DNA synthesis, but PDGF opposed the stimulatory effect of IGF I on collagen synthesis and IGF I prevented the PDGF effect on collagen degradation. In conclusion, PDGF stimulates calvarial DNA synthesis which causes an increased number of collagen-synthesizing cells, but PDGF also enhances bone collagen degradation.

Animals↗
PubMed1989

Analysis of hydroxyproline by high performance liquid chromatography and its application to collagen turnover studies in bone cultures.

We describe a high performance liquid chromatography (HPLC) technique for separating and quantitating hydroxyproline in calvarial cultures. Using a reverse-phase Nova-Pak C18 column and a 140 mM sodium acetate, 0.05% triethylamine (TEA), 6% acetonitrile solvent system, we obtained a complete separation of hydroxyproline. Recovery of added standards ranged from 89 to 103% and intraassay variability was less than 8%. [3H]hydroxyproline measurements were used to examine changes in collagen turnover in rat calvariae labeled with [3H]proline and "chased" in the presence of 10 mM unlabeled proline. The addition of parathyroid hormone (PTH) during a 24-48 hour "chase" period increased the release of acid-soluble [3H]hydroxyproline into the culture medium, indicating an increase of fully degraded collagen. This method offers a sensitive and reproducible technique for monitoring changes in bone matrix degradation and in studying agents that modify this process.

Animals↗
PubMed1989

Insulin-like growth factor I mediates selective anabolic effects of parathyroid hormone in bone cultures.

PTH was studied for its effects on bone formation in cultured rat calvariae. 0.01-10 nM PTH stimulated [3H]thymidine incorporation into DNA by up to 4.8-fold. Although continuous treatment with PTH for 24-72 h inhibited [3H]proline incorporation into collagen, transient (24 h) treatment enhanced [3H]proline incorporation into collagen 24-48 h after the hormone was removed. The collagen stimulated by PTH was type I and the effect was observed in the periosteum-free bone and was not blocked by hydroxyurea. Furthermore, treatment with 1-100 nM PTH for 24 h increased insulin-like growth factor (IGF) I concentrations by two to fourfold, and an IGF I antibody prevented the PTH stimulation of collagen synthesis, but not its mitogenic effect. In conclusion, continuous treatment with PTH inhibits calvarial collagen, whereas transient treatment stimulates collagen synthesis, and the stimulatory effect is mediated by local production of IGF I.

Animals↗
PubMed1989

Regulatory effects of insulin-like growth factors I and II on bone collagen synthesis in rat calvarial cultures.

Insulin-like growth factors I and II (IGF-I and -II) are polypeptides secreted by skeletal cells and are considered regulators of bone formation. IGF-I and -II were studied for their effects on collagen synthesis and degradation in cultures of intact fetal rat calvariae and on type I collagen transcript levels in osteoblast-enriched (Ob) cells from fetal rat parietal bone. IGF-I and -II increased [3H]proline incorporation into type I collagen independently of their effect on cell replication. IGF-I and -II also decreased collagen degradation in calvarial cultures. Both factors had similar actions, although IGF-I stimulated collagen synthesis at 10 nM, and IGF-II at 30 nM. In Ob cells, IGF-I and -II also increased [3H]proline incorporation into type I collagen, but the effect was seen at 100 nM, and neither factor decreased collagen degradation. Slot blot analysis of IGF-I- and IGF-II-treated cells, using a rat type I collagen cDNA probe, revealed an increase in type I collagen transcripts. In conclusion, IGF-I and -II increase bone collagen synthesis and decrease collagen degradation in cultures of intact calvariae; the effect on collagen synthesis correlates with an increase in transcript levels in Ob cells.

Animals↗
PubMed1989

Parathyroid hormone enhances the transcript and polypeptide levels of insulin-like growth factor I in osteoblast-enriched cultures from fetal rat bone.

PTH stimulates bone resorption and formation, but the mechanism of its anabolic effect is unknown. The effects of PTH on bone formation could be mediated by local regulators, either by altering their binding to receptors or by modulating their synthesis. Cell extracts from PTH-treated osteoblast-enriched cultures isolated from fetal rat parietal bones were examined by Northern blot analysis for changes in mRNAs encoding insulin-like growth factor I (IGF-I), transforming growth factor-beta, and beta 2-microglobulin. PTH did not influence transforming growth factor-beta or beta 2-microglobulin transcript levels. In contrast, PTH-(1-34) had a biphasic stimulatory effect on IGF-I transcript levels; 0.1-10 nM PTH increased IGF-I transcripts by 100-200% after a 6-h treatment, while 100 nM PTH induced a 100% increase. In addition, PTH at 0.01-10 nM increased immunoreactive IGF-I (iIGF-I) in culture medium by 40-200% at 24 h. Maximal increases in IGF-I transcripts occurred at 6 h, while iIGF-I accumulated throughout 24 h of culture. These results are compared to the effects of 0.5-50 nM GH, which increased IGF-I transcripts by 30% and iIGF-I by 50-100%. Therefore, PTH enhanced local IGF-I synthesis by increasing IGF-I transcripts, and this effect may in part mediate the anabolic actions of PTH on bone.

Animals↗
PubMed1989

Platelet-derived growth factor enhances deoxyribonucleic acid and collagen synthesis in osteoblast-enriched cultures from fetal rat parietal bone.

Platelet-derived growth factor (PDGF) or closely related proteins are found in bone matrix and are produced by cultured osteosarcoma cells. In serum-deprived osteoblast-enriched (ob) cultures from fetal rat bone, recombinant human PDGF (composed of a B chain homodimer) at 0.1-3 nM enhanced the rate of DNA synthesis by 2- to 8-fold within 24 h of treatment, and 0.3-3 nM PDGF increased cell number by 1.3- to 1.6-fold. Unlike results with rat kidney fibroblast cultures, the mitogenic effect of PDGF in ob cultures was not synergistic with that of insulin-like growth factor I. PDGF at 0.3-10 nM also enhanced the rates of collagen and noncollagen protein synthesis in ob cultures by 1.5- to 4.0-fold, and these increases were blocked when DNA synthesis was prevented. The stimulatory effects of PDGF did not appear specific to ob cultures from fetal rat bone, since similar increases were found in bone cell cultures containing fibroblasts and osteoblast precursors. PDGF binding at 4 C to ob cultures indicated a single class of receptors with a Kd of 0.16 nM and approximately 60,000 sites/cell. Polyacrylamide gel of 125I-PDGF bound and cross-linked to ob cultures revealed a single radioactive band at approximately 180,000-190,000 mol wt. The present studies, therefore, indicate that PDGF can directly increase replication and matrix protein synthesis by both differentiated and undifferentiated bone cells, and that bone- or platelet-derived PDGF may have an important anabolic role in bone remodeling or fracture repair.

Animals↗
PubMed1989

Parathyroid hormone-related protein modulates the effect of transforming growth factor-beta on deoxyribonucleic acid and collagen synthesis in fetal rat bone cells.

Proteins with biochemical function and sequence similarity to PTH are produced by many tumors associated with hypercalcemia and may have a role in pathological bone remodeling. Synthetic polypeptides comprising the amino-terminus of human PTH-related protein (PTH-rp) were examined for effects in intact fetal rat calvariae, and in osteoblast-enriched (ob) cultures isolated from fetal rat parietal bone. In cultured calvariae, 0.5-5 nM PTH-rp stimulated [3H]thymidine incorporation into DNA by 25-70% after 24 h of treatment and decreased relative [3H]proline incorporation into collagen by 50%; the inhibitory effect on collagen production was not altered by hydroxyurea, which decreased DNA synthesis by 85%. PTH-rp also increased [3H]hydroxyproline levels by 100% in culture medium from bones prelabeled with [3H]proline, indicating accelerated matrix turnover. In contrast to results in intact calvariae, PTH-rp had little effect on basal DNA and collagen synthesis in serum-deprived ob cultures. However, when ob cultures were treated with transforming growth factor type beta at concentrations similar to those found in calvarial culture medium, 0.02-2 nM PTH-rp enhanced DNA synthesis and decreased collagen production. Furthermore, equimolar PTH-rp and PTH concentrations similarly displaced 125I-PTH-rp binding and enhanced cAMP synthesis in ob cultures. These studies suggest that PTH-rp regulates osteoblastic cell activity primarily through PTH-related pathways and may act in part by modulating the effects of locally produced transforming growth factor-beta in bone.

Animals↗
PubMed1989

Effects of fibroblast growth factors on deoxyribonucleic acid and collagen synthesis in rat parietal bone cells.

Acidic fibroblast growth factor (aFGF) and basic FGF (bFGF) are related molecules that are extractable from bone matrix and may be important in the maintenance of normal bone physiology. The influence of each agent on DNA and protein synthesis was studied using bone-derived primary cell cultures. Both forms of FGF were relatively more mitogenic for bone cell populations with fewer osteoblastic (Ob) characteristics than for Ob-enriched cultures. However, in the Ob cultures, bFGF was intrinsically 10-fold more stimulatory than aFGF, whereas heparin enhanced the mitotic response only to aFGF. An optimal dose of either aFGF or bFGF (100 ng/ml) decreased alkaline phosphatase activity and increased the rate of noncollagen and collagen protein synthesis in Ob cultures. The stimulatory effect was relatively greater on noncollagen than on collagen synthesis, which resulted in a decrease in percent collagen synthesis. Neither factor altered the rate of collagen degradation. Furthermore, hydroxyurea diminished, but did not prevent, the stimulatory effect of each factor on rates of protein synthesis. In contrast, polyacrylamide gel analysis of newly synthesized protein and Northern blot analysis of steady state alpha 1 type I procollagen mRNA indicated differential effects by each agent on procollagen synthesis and processing. These studies suggest that the FGFs may produce their effects on Ob cells through both shared and disparate mechanisms, with the net result being a decrease in the expression of the osteoblastic phenotype.

Animals↗
PubMed1989

Effects of transforming growth factors on bone cells.

Bone formation results from the anabolic and catabolic functions of osteoblasts and osteoclasts within bone. The activities of these cell populations are controlled by complex interacting effects generated by local (bone-derived) and systemic (hormone) growth regulators. One of the more abundant growth regulators produced by bone cells and associated with bone matrix is transforming growth factor beta (TGF-beta). Recent studies indicate that TGF-beta controls the abundance and the biochemical function of osteoblasts and osteoclasts. Also, both TGF-beta production by bone cells, and its effects on bone cell activity, can be influenced by other local growth factors and osteotropic hormones.

Animals↗
PubMed1989

Insulin-like growth factor (IGF) and bone.

Bone is not only a rich source of a diverse group of growth factors, but is also a very responsive tissue to such growth promoting agents. IGF-I and IGF-II are reported to be synthesized and retained in bone. While both IGF-I and IGF-II stimulate DNA, collagen, and noncollagenous protein synthesis in cultured calvariae, these explant cultures have quantitative differential sensitivities to these IGF's. In addition to the observed increase in collagen synthesis, collagen degradation decreased in calvariae treated with IGF-I or IGF-II.

Animals↗
PubMed1989

The role of growth factors in skeletal remodeling.

Bone remodeling is a complex process regulated by systemic agents and local factors. During the past several years most or perhaps all the growth factors associated with the bone matrix have been characterized, and important information regarding hematologic factors has become available. Growth factors have significant effects on bone remodeling and likely play a major role in the maintenance of normal bone mass, wound healing, and fracture repair (Tables 3 and 4). Current studies indicate that systemic hormones regulate the synthesis and receptor binding of a number of local factors. By these mechanisms, specific hormones may target their effects to the skeleton. Although our knowledge of local growth factors is still limited, future work should determine their role in skeletal physiologic and pathophysiologic conditions and their potential as therapeutic agents.

Animals↗
PubMed1988

Further biochemical and molecular characterization of primary rat parietal bone cell cultures.

Primary bone cell cultures are used widely to examine the regulation of bone metabolism by growth factors and hormones. Characterization of this model system is now being conducted at the molecular level to define modulation of gene expression. Cells were obtained from rat parietal bone by sequential collagenase digestions. Cell populations were evaluated for bone-related products, including collagen isoform expression and mRNA levels, alkaline phosphatase activity, and osteocalcin production. Serum-deprived, confluent cultures of the first and second collagenase-released populations produced a lower percentage of total protein as collagen than the third, fourth, and fifth populations, while co-culturing the third through fifth populations resulted in the highest level. Collagen typing on SDS-polyacrylamide gels revealed an abundance of mature type I collagen in all cell populations; type III collagen synthesis was undetectable by this method. This is in contrast to the presence of cytoplasmic mRNA for both type I and type III collagen in all cell populations, suggesting post-transcriptional modulation of type III collagen synthesis. The expression of alkaline phosphatase and osteocalcin was highest in cultures of later released cells, indicating that these cell populations display phenotypic characteristics associated with cells of the osteoblast lineage.

Alkaline Phosphatase↗
PubMed1988

Isolation of growth factors from adult bovine bone.

Fetal rat calvariae synthesize transforming growth factor beta (TGF beta), beta 2 microglobulin (beta 2 m), and insulinlike growth factor I (IGF I), but, except for TGF beta, it is not known if these polypeptides are also present in adult bone tissue. Pulverized bovine bone, extracted with 0.5 N HCl and 4 M guanidine HCl and fractionated by gel filtration, was found to contain several biologically active components when tested for its effects on DNA synthesis in osteoblast-rich cell cultures. TGF beta, beta 2 m, and IGF I were identified and further purified using high performance liquid chromatography (HPLC). TGF beta, identified by a standard TGF beta bioassay or by immunoreactivity, was purified by muBondapak C18 and muBondapak CN reversed phase HPLC. beta 2 m, identified by immunoreactivity, required an additional fractionation step on a DEAE-HPLC column for complete purification. IGF I, identified by immunoreactivity, was purified by HPLC using a muBondapak C18 and a DEAE-HPLC column. Purified TGF beta, beta 2 m, and IGF I migrated as single bands on polyacrylamide gel electrophoresis with respective molecular masses of 24,000, 10,000, and 7,500. In conclusion, adult bone matrix, like fetal bone cultures, contains TGF beta, beta 2 m, and IGF I and these factors may play a role in adult skeletal remodeling.

Animals↗
PubMed1988

Effects of bone associated growth factors on DNA, collagen and osteocalcin synthesis in cultured fetal rat calvariae.

Studies in bone and bone cell cultures have shown that osteocalcin synthesis is dependent on the maturity of the osteoblast and the presence of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3. The bone matrix is a rich source of growth factors that play a role in bone formation, but their effects on osteocalcin synthesis and their interactions with 1,25(OH)2D3 have not been examined. Insulin-like growth factor I (IGF I), basic and acidic fibroblast growth factor (bFGF and aFGF), platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF beta), are growth factors associated with the bone matrix. These factors were shown to stimulate [3H]thymidine incorporation into DNA in 24 h cultures of fetal rat calvariae, and their effect was not modified by 1,25(OH)2D3. IGF I and TGF beta stimulated [3H]proline incorporation into calvarial collagen while the other growth factors studied did not; 1,25(OH)2D3 inhibited collagen synthesis in control as well as in IGF I and TGF beta treated calvariae. IGF I, bFGF and aFGF stimulated osteocalcin synthesis 1.5 to 2.5 fold but only IGF I was synergistic with the stimulatory effect of 1,25(OH)2D3. PDGF and TGF beta had no effect on osteocalcin synthesis. In conclusion, bone matrix-associated factors have important mitogenic effects in bone cultures, but only IGF I and FGFs stimulate osteocalcin synthesis, an effect that is of small magnitude when compared to that of 1,25(OH)2D3.

Animals↗
PubMed1988

Parathyroid hormone modulates transforming growth factor beta activity and binding in osteoblast-enriched cell cultures from fetal rat parietal bone.

Transforming growth factor beta (TGF-beta) is produced by bone cells, is abundant in bone matrix, and regulates bone cell biochemical processes. In osteoblast-enriched fetal rat parietal bone cell cultures, low TGF-beta doses increase DNA synthesis, whereas higher levels are less mitogenic, stimulate collagen production, and decrease alkaline phosphatase activity. Parathyroid hormone by itself has minimal effects on these processes, but it opposes the effects of TGF-beta and alters TGF-beta binding to its receptors in osteoblast-enriched cultures. Some functions ascribed to parathyroid hormone in bone may therefore result from alterations in TGF-beta activity, suggesting that the local effects of TGF-beta in bone are under systemic hormonal control.

Animals↗