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Biomedical subjects

E Canalis

Publications and source records attributed to E Canalis.

At least 109 records · Page 6Linked to original sources
PubMed1992

Isoform-specific regulation of platelet-derived growth factor activity and binding in osteoblast-enriched cultures from fetal rat bone.

In osteoblast-enriched cultures from fetal rat bone, the A-chain homodimer of platelet-derived growth factor (PDGF-AA) is less potent than the PDGF isoforms containing B chain subunits (PDGF-AB and PDGF-BB), but normal osteoblasts appear to synthesize only PDGF-A subunit mRNA and polypeptide. However, other agents may regulate PDGF-AA activity in skeletal tissue. Pretreatment of osteoblast-enriched cultures with interleukin 1 alpha (IL-1 alpha) or tumor necrosis factor-alpha (TNF-alpha) synergistically enhanced the mitogenic effect of PDGF-AA coincident with increased binding site occupancy, but neither factor augmented PDGF-BB activity or binding. Polyacrylamide gel analysis showed 125I-PDGF-AA binding complexes predominantly at greater than 200 kD and faint labeling at 185 kD. After IL-1 alpha or TNF-alpha pretreatment, PDGF-AA binding increased at both sites, but this effect was more striking at 185 kD, which co-migrated with 125I-PDGF-BB-labeled complexes. PDGF-AA binding sites were rapidly lost by comparison to those for PDGF-BB in cycloheximide-treated cultures, but they remained relatively enhanced by IL-1 alpha and TNF-alpha pretreatment. These studies indicate that IL-alpha and TNF-alpha increase PDGF-AA binding and activity for osteoblasts by mechanisms that are at least in part independent of new receptor synthesis, and suggest regulatory events that could control how PDGF binding sites specifically recognize different ligands.

Animals↗
PubMed1992

Constitutive synthesis of insulin-like growth factor-II by primary osteoblast-enriched cultures from fetal rat calvariae.

While a number of osteotropic hormones regulate insulin-like growth factor-I (IGF-I) synthesis in osteoblast-enriched (Ob) and intact bone cultures, their direct effects on IGF-II production are still unresolved. For example, cAMP stimulators, such as PTH and prostaglandin E2, increase Ob IGF-I transcript and polypeptide levels within the first 24 h of treatment, but have no effect on IGF-II expression. To examine the possibility that other circulating factors could directly modify IGF-II synthesis by osteoblasts, primary rat Ob cultures were briefly treated with a number of polypeptide and steroid hormones known to regulate bone metabolism. Prepro-IGF-II steady state transcripts were assessed by Northern blot analysis, and immunoreactive polypeptide levels (iIGF-II) were examined by RIA. Predominant prepro-IGF-II transcripts of 3.7 kilobases were readily detected in quiescent Ob cultures, and constitutive iIGF-II levels were approximately 2-7 nM throughout the first 24 h of culture. GH, placental lactogen, insulin, cortisol, testosterone, T3, 17 beta-estradiol, and 1,25-dihydroxyvitamin D3 each had no effect on prepro-IGF-II transcripts within 6 h or on iIGF-II polypeptide expression within a 24-h period. These studies indicate that IGF-II synthesis is constitutive in unstimulated primary fetal rat Ob cultures, and that these levels are not directly modulated by short term treatment with a variety of osteotropic hormones.

Animals↗
PubMed1992

Determination and expression of platelet-derived growth factor-AA in bone cell cultures.

We describe a sensitive technique for the extraction and quantitation of platelet-derived growth factor (PDGF)-AA in serum-free culture medium conditioned by fetal rat osteoblast-enriched (Ob) cells and demonstrate the expression of PDGF-A mRNA in Ob cells. Using C18 Sep-Pak chromatography with a methanol step gradient, we extracted immunoreactive PDGF-AA from the culture medium. A RIA protocol employing a recombinant human PDGF-AA standard enabled us to measure picomolar equivalents of PDGF-AA in Ob cell culture medium. The recovery of recombinant human PDGF-AA was 50 +/- 4%, and the coefficient of variation, including chromatographic extraction, was 15% for intraassay and 8.3% for interassay variability. The polyclonal antibody to recombinant human PDGF-AA displayed approximately 20-30% cross-reactivity with PDGF-AB, but did not bind PDGF-BB or other growth factors and cytokines known to be secreted by bone cells. Medium from Ob cells cultured for 24 h contained 0.9-1.3 pM human PDGF-AA equivalents, and exposure to cycloheximide (3.6 microM) decreased those levels by 65%. Treatment of cells with recombinant human transforming growth factor-beta 1 at 0.04-4 nM for 24 h increased PDGF-AA levels by up to 3.5-fold. Northern blot analysis of RNA from Ob cells revealed the expression of PDGF-A, but not PDGF-B, transcripts, and transforming growth factor-beta 1 at 0.04 and 0.2 nM increased steady state PDGF-A mRNA by 3- to 6-fold. Our studies describe a sensitive and reproducible technique for monitoring PDGF-AA levels in cell-conditioned medium and demonstrate that Ob cells synthesize PDGF-AA.

Animals↗
PubMed1992

Role of platelet derived growth factor in bone cell function.

PDGF is a mitogen for cells of the osteoblastic lineage. PDGF is present in the systemic circulation and is locally synthesized by skeletal cells. The systemic form primarily contains PDGF B chains, which are intrinsically more active than PDGF A subunits, the forms secreted by normal bone cells. PDGF AA is regulated by other growth factors and cytokines, which modulate its binding to osteoblastic receptors and its synthesis by skeletal cells. The exact role of PDGF in bone remodelling is still uncertain and current information suggests that this factor has a function in the response to inflammation and wound healing.

Animals↗
PubMed1991

Relative binding and biochemical effects of heterodimeric and homodimeric isoforms of platelet-derived growth factor in osteoblast-enriched cultures from fetal rat bone.

Platelet-derived growth factor (PDGF) exists as a homodimer or a heterodimer comprising either PDGF-A or PDGF-B subunits, and each isoform occurs in various tissues, including bone. Although the stimulatory effects of PDGF-BB have been studied in cultures of bone cells and intact bone fragments, the influence of other isoforms that may arise locally or systematically in vivo, has not been reported. Therefore recombinant human PDGF-BB, PDGF-AB, and PDGF-AA were evaluated in osteoblast-enriched cultures from fetal rat bone. Within 24 hours these factors produced a graded response in bone cell DNA and protein synthesis, with half-maximal effects at approximately 0.6, 2.1, and 4.8 nM PDGF-BB, PDGF-AB, and PDGF-AA, respectively. Increases in collagen and noncollagen protein synthesis were abrogated when DNA synthesis was blocked with hydroxyurea. Furthermore, each factor reduced alkaline phosphatase activity, PDGF-BB being the most inhibitory. Binding studies with 125I-PDGF-BB or 125I-PDGF-AA and each unlabeled PDGF isoform produced discrete ligand binding and displacement patterns: 125I-PDGF-BB binding was preferentially displaced by PDGF-BB (Ki approximately 0.7 nM), less by PDGF-AB (Ki approximately 2.3 nM) and poorly by PDGF-AA. In contrast, 125I-PDGF-AA binding was measurably reduced by PDGF-AA (Ki approximately 4.0 nM), but was more effectively displaced by PDGF-BB or PDGF-AB (each with Ki approximately 0.7 nM). These studies indicate that each PDGF isoform produces biochemical effects proportional to binding site occupancy and suggest that receptors that favor PDGF-B subunit binding preferentially mediate these results in osteoblast-enriched bone cell cultures.

Alkaline Phosphatase↗
PubMed1991

Activin-A binding and biochemical effects in osteoblast-enriched cultures from fetal-rat parietal bone.

Activin, a disulfide-linked polypeptide dimer first isolated from gonadal tissue extracts, has amino acid sequence and structural homology with transforming growth factor beta (TGF beta). Along with other activities, TGF beta regulates replication and differentiation and interacts with a defined set of binding sites on isolated bone cells. To determine if activin shares these properties, recombinant human activin-A (A-chain homodimer) was examined in osteoblast-enriched cultures obtained from fetal-rat parietal bone. After 23 h of treatment, 60 to 6,000 pM activin-A increased the rate of [3H]thymidine incorporation into DNA 1.5- to 4.0-fold, and at 600 to 6,000 pM, it enhanced the rate of [3H]proline incorporation into collagen and noncollagen protein by up to 1.7-fold. Like earlier studies with TGF beta in primary osteoblast-enriched cultures, the stimulatory effects of activin-A on DNA and protein synthesis were opposed by parathyroid hormone, and the influence of activin-A on collagen synthesis was independent of cell replication. Binding studies with 125I-activin-A indicated approximately 8,000 high-affinity (Kd = 0.4 nM) and 300,000 low-affinity (Kd = 40 to 50 nM) binding sites per cell. Polyacrylamide gel analysis revealed 125I-activin-A-binding complexes of Mr greater than 200,000 and 73,000 which did not appear to correspond to primary TGF beta-binding sites. These results indicate that activin-A produces TGF beta-like effects in bone and that some of these effects may be mediated, at least in part, by distinct activin receptors on bone cells.

Activins↗
PubMed1991

Glucocorticoid regulation of transforming growth factor beta 1 activity and binding in osteoblast-enriched cultures from fetal rat bone.

Transforming growth factor beta (TGF-beta) enhances replication and bone matrix protein synthesis and associates with distinct binding sites in osteoblast-enriched cultures from fetal rat bone. In the organism high levels of or sustained exposure to glucocorticoids alters bone cell activity and decreases bone mass, effects that may be mediated in part by changes in local TGF-beta actions in skeletal tissue. Preexposure of osteoblast-enriched cultures to 100 nM cortisol reduced the stimulatory effects of TGF-beta 1 on DNA and collagen synthesis by 40 to 50%. Binding studies showed that cortisol moderately enhanced total TGF-beta 1 binding, but chemical cross-linking and polyacrylamide gel electrophoretic analysis revealed an increase only within Mr 250,000 (type III) TGF-beta-binding complexes, which are thought to represent extracellular TGF-beta storage sites. In contrast, a decrease in TGF-beta 1 binding was detected in Mr 65,000 (type I) and 85,000 (type II) complexes, which have been implicated as signal-transducing TGF-beta receptors. Our present studies therefore indicate that glucocorticoids can decrease the anabolic effects of TGF-beta 1 in bone, and these may occur in part by a redistribution of its binding toward extracellular matrix storage sites. Alterations of this sort could contribute to bone loss associated with glucocorticoid excess.

Animals↗
PubMed1991

Growth factors and cytokines in bone cell metabolism.

Growth factors regulate the growth and differentiated function of cells. Skeletal cells synthesize fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor, transforming growth factor beta, and additional cytokines. Some of the growth factors produced by bone cells primarily stimulate bone cell replication, whereas others also affect the differentiated function of the osteoblast. Skeletal growth factors also may play a role in the pathogenesis and therapy of metabolic bone disease.

Bone Development↗
PubMed1991

Prostaglandin E2 stimulates insulin-like growth factor I synthesis in osteoblast-enriched cultures from fetal rat bone.

Prostaglandin E2 (PGE2) affects both bone resorption and formation, but its mechanism of action remains unclear. PGE2 is known to elevate intracellular cAMP levels in a variety of culture systems. Agents that increase cAMP in primary osteoblast-enriched (Ob) cultures enhance the synthesis of skeletal insulin-like growth factor I (IGF-I), a potent anabolic factor for bone. A 5 min exposure to PGE2 at 0.01-1 microM enhanced cAMP synthesis in Ob cultures by 8- to 54-fold, and within 6 h produced up to a 3- fold increase in steady state prepro-IGF-I transcripts. The stimulatory effect of PGE2 on IGF-I messenger RNA was first evident within 4 h of treatment and remained elevated for at least 24 h. Furthermore, 0.01-1 microM PGE2 increased immunoreactive IGF-I polypeptide accumulation by 1.9- to 4.7-fold. In contrast PGE2 did not elevate steady state IGF-II mRNA or polypeptide levels within this time frame. Although PTH increase cAMP, intracellular calcium, and PGE2 production by bone cells, our previous studies indicate that the stimulatory effect of PTH on IGF-I production is cAMP, but not calcium dependent. Inhibition of PGE2 synthesis by exposure to indomethacin did not alter basal or PTH-stimulated IGF-I levels, substantiating that the effect of PTH on IGF-I is not PGE2 dependent. These studies indicate that PGE2 production, a feature common to many agents that enhance bone resorption, could contribute to the coupling of bone resorption and new bone formation, by way of its ability to increase cAMP and, consequently, IGF-I synthesis by the osteoblast.

Animals↗
PubMed1991

Effects of desamino-(1-3)-insulin-like growth factor I on bone cell function in rat calvarial cultures.

Insulin-like growth factor (IGF) I, a polypeptide synthesized by skeletal cells, and its amino terminus truncated derivative desamino-(1-3)-IGF I (des-IGF I) were compared for their effects on bone formation in vitro. Des-IGF I and IGF I were studied for their effects on DNA and collagen synthesis in cultures of intact fetal rat calvariae and of osteoblast-enriched (Ob) cells from fetal rat parietal bone, and for their ability to bind to IGF receptors in Ob cells and to IGF binding proteins (IGF-BPs) from calvariae. Des-IGF I and IGF I increased [3H] thymidine incorporation into DNA, [3H]proline incorporation into collagen and noncollagen protein, and the mitotic index in intact calvariae. Both factors had similar actions in calvariae. Des-IGF I stimulated all parameters studied at 1 nM, and IGF I was effective on the labeling of DNA at 1 nM, but concentrations of 10 nM were required to observe changes in collagen and noncollagen protein synthesis and in the mitotic index. The effect of des-IGF I on collagen synthesis was independent from that on DNA synthesis, as it is known for IGF I, and both forms of IGF I were equally potent for their inhibitory effects on collagen degradation in calvarial cultures. In Ob cells, neither des-IGF I nor IGF I altered the incorporation of [3H]thymidine into DNA, but both factors at 10-100 nM increased [3H]proline incorporation into collagen to a similar extent. Receptor studies revealed a similar binding capacity for des-IGF I and IGF I to the IGF I receptor(s) in Ob cells, although at 0.2 nM des-IGF I was slightly more effective than IGF I. In contrast, des-IGF I was 100-fold less effective than IGF I for its ability to bind to partially purified IGF-BPs from cultured calvariae. In conclusion, des-IGF I enhances calvarial DNA and collagen synthesis and osteoblastic collagen synthesis to a somewhat greater extent than IGF I, in spite of a much lesser affinity for IGF-BPs.

Animals↗
PubMed1991

Regulation of insulin-like growth factor-II production in bone cultures.

Although bone matrix is a rich source of insulin-like growth factor-II (IGF-II), little is known about the regulation of its synthesis by bone cells. This is due in part to the lack of simple and reliable assays to measure IGF-II. We have developed a method to dissociate IGF-II from its binding proteins by acidification and ultrafiltration, and quantitated IGF-II by RIA in 24- to 72-h cultures of 21-day-old fetal rat calvariae. The coefficient of variation of the assay was 13.8% or less; the recovery of IGF-II was 30-50%, and IGF-I cross-reacted 1% or less in the assay compared to IGF-II standards. The IGF-II concentrations in calvarial culture medium were in the 1- to 3-nM range, and these levels were suppressed by cycloheximide (3.6 microM) by almost 80%. Continuous treatment with placental lactogen, PTH, GH, insulin, or T3 did not modify IGF-II concentrations in 24- to 72-h cultures. Treatment with 17 beta-estradiol, testosterone, and 1,25-dihydroxyvitamin D3 also had no effect on IGF-II levels, whereas cortisol (10-100 nM) decreased IGF-II concentrations by 20-50%. Transforming growth factor-beta, prostaglandin E2, and platelet-derived growth factor BB did not alter IGF-II levels, and basic fibroblast growth factor (0.06-6 nM) for 72 h decreased calvarial IGF-II by 30-50%. In conclusion, 21-day-old fetal rat calvariae secrete IGF-II, and its concentration in culture medium is decreased by cortisol and basic fibroblast growth factor.

Animals↗
PubMed1990

Cyclic AMP induces insulin-like growth factor I synthesis in osteoblast-enriched cultures.

Earlier studies indicate that parathyroid hormone (PTH) enhances insulin-like growth factor I (IGF-I) synthesis in primary osteoblast-enriched fetal rat cell cultures and the stimulatory effect of PTH on bone collagen synthesis is mediated at least in part by IGF-I. Cyclic AMP (cAMP) is a second messenger for signal transduction by PTH to its target cells, although calcium may also serve this function. We now demonstrate that isobutylmethylxanthine, forskolin, and dibutyryl cAMP, agents that elevate intracellular cAMP levels by discrete mechanisms, also enhanced the steady state transcript and polypeptide level of IGF-I in osteoblast-enriched cultures. The calcium ionophore ionomycin and phorbol myristate acetate did not increase IGF-I synthesis. In contrast, none of the agents tested increased the steady state transcript or polypeptide levels for IGF-II. The rat IGF-I gene is greater than 90 kilobases in length, and contains at least three promoter regions. Our present data represent the first demonstration of cAMP mediated IGF-I gene regulation and indicate the potential for preferential promoter usage for modulating IGF-I gene expression in bone.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Recombinant transforming growth factor type beta 3: biological activities and receptor-binding properties in isolated bone cells.

We have recently cloned the cDNA for transforming growth factor type beta 3 (TGF-beta 3), a new member of the TGF-beta gene family. We examined the biological effects of recombinant TGF-beta 3 protein in osteoblast-enriched bone cell cultures. In this report we demonstrate that TGF-beta 3 is a potent regulator of functions associated with bone formation, i.e., mitogenesis, collagen synthesis, and alkaline phosphatase activity. In a direct comparison between TGF-beta 3 and TGF-beta 1, TGF-beta 3 appeared to be three- to fivefold more potent than TGF-beta 1. Our cross-linking experiments with iodinated TGF-beta showed that in osteoblast-enriched bone cell cultures, both TGF-beta 3 and TGF-beta 1 associated with the same three cell surface binding sites. Scatchard analysis of receptor competition studies indicated the presence of high-affinity binding sites for TGF-beta 3 in the picomolar range. TGF-beta 3 showed an approximately fourfold-higher apparent affinity than TGF-beta 1 in overall binding.

Alkaline Phosphatase↗
PubMed1990

Receptors for insulin-like growth factors-I and -II in osteoblast-enriched cultures from fetal rat bone.

Insulin-like growth factors-I and -II (IGF-I and IGF-II) are produced by bone cells and stored in bone matrix, and stimulate bone cell DNA synthesis and type I collagen production. Earlier studies in cells from an assortment of tissues indicate that IGF-I binds to membrane receptors of various relative molecular mass (Mr), whereas IGF-II binds predominantly to the mannose-6-phosphate transferase. In the present studies we have examined the IGF receptor profile in osteoblast-enriched cultures prepared from fetal rat parietal bone. Scatchard binding kinetics with either 125I-IGF-I or 125I-IGF-II were curvilinear, indicating high and low affinity receptor classes for each ligand. Chemical cross-linking and polyacrylamide gel analysis revealed that 125I-IGF-I bound at Mr 130,000, 240,000, and 260,000, whereas 125I-IGF-II bound predominantly at Mr 240,000. Unlabeled IGF-I displaced 125I-IGF-I with high affinity at Mr 260,000 and 130,000 and with lower affinity at Mr 240,000. Unlabeled IGF-II preferentially displaced either radioactive ligand at Mr 240,000, but at high concentrations displaced 125I-IGF-I at all binding sites. Furthermore, mannose-6-phosphate enhanced binding by both 125I-IGF-I and 125I-IGF-II at Mr 240,000, and antibody to the rat IGF-II receptor prevented binding only at this location. The relative affinities of IGF-I and IGF-II for the primary IGF-I receptor correlate with their anabolic activities in bone cultures and suggest that the IGF-I receptor mediates the growth-promoting effects of both agents in bone.

Animals↗
PubMed1990

Transforming growth factor-beta stimulates bone matrix apposition and bone cell replication in cultured fetal rat calvariae.

Transforming growth factor-beta (TGF beta) stimulates the expression of extracellular matrix proteins and may be a local regulator of bone growth. The aims of this research were to localize the effect of TGF beta on bone matrix formation and to determine if this effect was dependent on increased cell replication, using histomorphometry and autoradiography of bone organ cultures. Half-calvariae of 21-day-old fetal rats were cultured with native or recombinant TGF beta 1 for 24 h and labeled either with [3H]proline for 0-24 or 24-48 h or with [3H]thymidine for the last 6 h of culture. Bones were fixed in glutaraldehyde, embedded in glycol methacrylate, and processed for autoradiography. Bone matrix formation was assessed as the matrix apposition rate per day and the percentage of [3H]proline-labeled bone surface. Cell replication was evaluated based on the number and percentage of [3H]thymidine labeled cells in the osteoblast cell zone, the osteoprogenitor cell zone, and the pericranial fibroblastic periosteum. Both native and recombinant TGF beta at 1-30 ng/ml increased bone matrix formation by 25-40% (P less than 0.05). At 30 ng/ml, TGF beta had a generalized mitogenic effect as cell replication increased by approximately 2-fold in all cell zones of the pericranial periosteum. TGF beta had specific effects on bone cell differentiation. The number of unlabeled cells lining the bone surface increased, and the number of osteoclasts on bone decreased. Inhibition of cell replication by hydroxyurea only partially blocked the stimulatory effect of TGF beta on bone matrix formation, suggesting that TGF beta may have independent effects on cell replication and differentiated bone cell function. In summary, TGF beta had a generalized mitogenic effect on the pericranial periosteum and specific stimulatory and inhibitory effects on bone cell differentiation and function.

Animals↗
PubMed1990

Cortisol inhibits the synthesis of insulin-like growth factor-I in skeletal cells.

Supraphysiological levels of cortisol inhibit bone cell replication and matrix synthesis, but its mechanism of action is unknown and could be secondary to an inhibition of local growth factor synthesis. These inhibitory effects of cortisol are the converse of the observed anabolic influences of the endogenously produced insulin-like growth factor-I (IGF-I); therefore, cortisol was examined for its effect on the production of IGF-I in osteoblast (Ob)- and fibroblast/preosteoblast-enriched cell cultures prepared from fetal rat parietal bone. Synthesis of IGF-I was monitored by Northern blot analysis to determine steady state IGF-I mRNA levels and by an IGF-I-specific RIA to quantitate polypeptide levels in acidified and fractionated culture medium. Cortisol at 100 nM decreased IGF-I transcript levels by 60% or more in Ob cultures within 6 h of treatment, and the concentration of immunoreactive IGF-I by 50% after 24 h; these effects were observed in the absence of a change in cellular DNA content. In Ob cultures, PTH at 10 nM increased IGF-I transcripts at 6 h and polypeptide levels at 24 h by 2.5- and 4.1-fold, respectively, and cortisol opposed this effect. The inhibitory effect of cortisol was not specific for the Ob cell population, since at 100 nM it also decreased IGF-I transcript and immunoreactive IGF-I levels in fibroblast/preosteoblast cultures and opposed the stimulation of IGF-I synthesis after treatment with 100 ng/ml GH. In conclusion, high levels of cortisol decrease skeletal IGF-I synthesis by reducing IGF-I transcript levels, and this effect probably contributes to the inhibitory influence of cortisol on bone formation.

Animals↗