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G A Rodan

Publications and source records attributed to G A Rodan.

At least 109 records · Page 6Linked to original sources
PubMed1991

Cell density-dependent decrease in cytoskeletal actin and myosin in cultured osteoblastic cells: correlation with cyclic AMP changes.

During bone development, osteoblasts form a contiguous layer along recently deposited osteoid and their morphology changes from fibroblast-like to cuboidal. In culture, similar changes occur with increased cell density. We examined the possible role of cyclic AMP in this process since cyclic AMP was reported to increase in fibroblasts with increased cell density and similar shape changes were seen in response to parathyroid hormone, which also increases cellular cyclic AMP in osteoblastic cells. Osteoblast-enriched rat calvaria cells were seeded at increasing density. The distribution between Triton X-100 extractable and nonextractable actin and myosin was estimated by polyacrylamide gel electrophoresis. Intracellular cyclic AMP was estimated by prelabeling the cellular ATP pool with 3H-adenine, followed by extraction and separation of 3H-cAMP by high-performance liquid chromatography. We found that osteoblastic cells contain about 40 pg actin and 5.3 pg myosin per cell. Around 60% of the actin and 70% of the myosin were in the nonextractable (crosslinked) form at cell densities of 10,000 to 50,000 cells per cm2. Above 50,000 cells/cm2, there was a cell density-dependent reduction in crosslinked actin and myosin and a concomitant increase in cellular cyclic AMP. A comparable rise in cyclic AMP, produced by incubation with phosphodiesterase inhibitors, and treatment with other agents that increase cyclic AMP produced a similar decrease in the level of cytoskeletal actin and myosin. Cytochalasin B treatment, through its effect on actin polymerization, produced similar changes in cell shape and cytoskeletal actin. The findings suggest that an elevation in intracellular cyclic AMP may play a role in the density-dependent changes in cell shape and microfilament organization observed in osteoblasts.

Actins↗
PubMed1991

Parathyroid hormone promotes the disassembly of cytoskeletal actin and myosin in cultured osteoblastic cells: mediation by cyclic AMP.

Parathyroid hormone (PTH) alters the shape of osteoblastic cells both in vivo and in vitro. In this study, we examined the effect of PTH on cytoskeletal actin and myosin, estimated by polyacrylamide gel electrophoresis of Triton X-100 (1%) nonextractable proteins. After 2-5 minutes, PTH caused a rapid and transient decrease of 50-60% in polymerized actin and myosin associated with the Triton X-100 nonextractable cytoskeleton. Polymerized actin returned to control levels by 30 min. The PTH effect was dose-dependent with an IC50 of about 1 nM, and was partially inhibited by the (3-34) PTH antagonist. PTH caused a rapid transient rise in cyclic AMP (cAMP) in these cells that peaked at 4 min, while the nadir in cytoskeletal actin and myosin was recorded around 5 min. The intracellular calcium chelator Quin-2/AM (10 microM) also decreased cytoskeletal actin and myosin, to the same extent as did PTH (100 nM). To distinguish between cAMP elevation and Ca++ reduction as mediators of PTH action, we measured the phosphorylation of the 20 kD (PI 4.9) myosin light chain in cells preincubated with [32P]-orthophosphate. The phosphorylation of this protein decreased within 2-3 min after PTH addition and returned to control levels after 5 min. The calcium ionophore A-23187 did not antagonize this PTH effect. Visualization of microfilaments with rhodamine-conjugated phalloidin showed that PTH altered the cytoskeleton by decreasing the number of stress fibers. These changes in the cytoskeleton paralleled changes in the shape of the cells from a spread configuration to a stellate form with retracting processes. The above findings indicate that the alteration in osteoblast shape produced by PTH involve relatively rapid and transient changes in cytoskeletal organization that appear to be mediated by cAMP.

Actins↗
PubMed1991

Immobilization-related bone loss in the rat is increased by calcium deficiency.

The object of this study was to investigate whether a calcium-deficient diet increases the bone loss produced by mechanical hypofunction (disuse) in the rat. Male Sprague-Dawley rats of approximately 150 g were placed on either a normal diet or a calcium-deficient diet. After 7 days, all rats underwent unilateral hind-limb immobilization by sciatic neurectomy and were sacrificed 30 hours, 72 hours, or 10 days postsurgery. Femora were ashed and the total mineral content (ash weight) was determined. Tibiae were embedded, sectioned, and stained. The metaphyseal secondary spongiosa and the diaphyseal cortical bone were subjected to histomorphometric analysis. Femoral length and serum calcium were not affected by calcium intake or immobilization. Serum parathyroid hormone levels were elevated in rats on the calcium-deficient diet compared to those on the normal diet. Calcium deficiency caused a significant reduction in femoral ash weight (20-35%), tibial cortical thickness (16-20%), and trabecular bone volume (TBV) (33-39%) at 72 hours and 10 days postsurgery. Additional loss of bone mass occurred in the immobilized limb compared to the contralateral intact limb of both dietary groups. This loss occurred earlier (30 hours postsurgery versus 72 hours) in the animals on a calcium-deficient diet and was larger compared to animals on a normal diet (10.6% versus 4.8% at 72 hours and 17.9% versus 12.45% at 10 days). The total bone loss induced by the combination of a calcium-deficient diet and immobilization in this experiment was estimated to equal 46% of femoral ash weight and 79% of tibial TBV.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Retinoic acid increases zif268 early gene expression in rat preosteoblastic cells.

In this study we demonstrate that retinoic acid (RA) increases the expression of transcription factor zif268 mRNA in primary cultures of fetal rat calvarial cells and in simian virus 40-immortalized clonal rat calvarial preosteoblastic cells (RCT-1), which differentiate in response to RA, but not in the more differentiated RCT-3 and ROS 17/2.8 cells. The increased expression of zif268 mRNA is rapid (maximal within 1 h), transient (returns to basal levels by 3 h), detectable at RA doses of 10(-12)M, and independent of protein synthesis. The relative stimulation of zif268 mRNA by RA was much larger than that of other early genes, including c-fos, c-jun, and junB. The rate of transcription of RA-stimulated RCT-1 cells, estimated by nuclear run-on assays, was elevated, suggesting that RA regulation of zif268 gene transcription was at least in part transcriptional. Moreover, RA stimulated the transcriptional activity of a Zif268CAT (chloramphenicol acetyltransferase) plasmid containing 632 bp of zif268 5' regulatory sequences in RCT-1 cells but not in the more differentiated RCT-3 cells. These in vitro data support the in vivo observations which localize zif268 and RA receptor-gamma transcripts to bone and cartilage during development, suggesting that both RA and zif268 may play a role in osteoblast differentiation.

Animals↗
PubMed1991

Bisphosphonate action. Alendronate localization in rat bone and effects on osteoclast ultrastructure.

Studies of the mode of action of the bisphosphonate alendronate showed that 1 d after the injection of 0.4 mg/kg [3H]alendronate to newborn rats, 72% of the osteoclastic surface, 2% of the bone forming, and 13% of all other surfaces were densely labeled. Silver grains were seen above the osteoclasts and no other cells. 6 d later the label was 600-1,000 microns away from the epiphyseal plate and buried inside the bone, indicating normal growth and matrix deposition on top of alendronate-containing bone. Osteoclasts from adult animals, infused with parathyroid hormone-related peptide (1-34) and treated with 0.4 mg/kg alendronate subcutaneously for 2 d, all lacked ruffled border but not clear zone. In vitro alendronate bound to bone particles with a Kd of approximately 1 mM and a capacity of 100 nmol/mg at pH 7. At pH 3.5 binding was reduced by 50%. Alendronate inhibited bone resorption by isolated chicken or rat osteoclasts when the amount on the bone surface was around 1.3 x 10(-3) fmol/microns 2, which would produce a concentration of 0.1-1 mM in the resorption space if 50% were released. At these concentrations membrane leakiness to calcium was observed. These findings suggest that alendronate binds to resorption surfaces, is locally released during acidification, the rise in concentration stops resorption and membrane ruffling, without destroying the osteoclasts.

Alendronate↗
PubMed1991

Functional estrogen receptors in osteoblastic cells demonstrated by transfection with a reporter gene containing an estrogen response element.

Although a small number of estrogen receptors (ER) were visualized in osteoblastic cells, and estradiol (E2) has some effects on osteoblasts in vitro, the direct action of E2 on osteoblasts has not been fully established. To determine the presence of functional ER in osteoblasts, we transfected cells with a plasmid containing the chloramphenicol acetyl transferase (CAT) reporter gene and the estrogen-responsive element (ERE) from the vitellogenin A2 gene. E2-dependent induction of CAT activity was determined 48 h after transient transfection and subsequent treatment with 10-100 nM 17 beta-E2. 17 beta-E2, but not 17 alpha-E2, dihydrotestosterone, or progesterone, induced CAT activity in a dose-dependent manner (up to 6-fold) in rat calvarial fraction-3, RCT-3, PyMS, and UMR-106 cells as well as in the human osteosarcoma cell line SaOS-2/B-10. In contrast, E2 had no effect on the induction of CAT activity in the preosteoblastic cell lines RCT-1 and TRAB-11, in the rat osteosarcoma cell line ROS 17/2.8, and in the fibroblastic cell lines BALB-c/3T3 and NRK. Over-expression of ER using a simian virus-40-based expression vector not only conferred or enhanced E2-dependent induction of CAT in all cell types, but augmented E2-dependent expression of insulin-like growth factor-I and E2-stimulated DNA synthesis in primary calvarial and PyMS osteoblastic cells, respectively. These data show the presence of low levels of functional endogenous ER in some, but not all, osteoblastic cells and suggest that the abundance of ER may be rate limiting in the action of E2 on these cells.

3T3 Cells↗
PubMed1991

Estradiol regulation of insulin-like growth factor-I expression in osteoblastic cells: evidence for transcriptional control.

Insulin-like growth factor-I (IGF-I) has anabolic effects on skeletal tissues, acting as both a systemic hormone and an autocrine/paracrine regulator of cellular function. We have previously reported that estradiol (E2) stimulation of rat osteoblast proliferation in vitro was inhibited by IGF-I antibodies. We show here that E2, similar to IGF-I, also increases alpha 1(I) procollagen mRNA levels in primary cultures of rat calvarial osteoblasts. The E2 effect on collagen mRNA lags behind that produced by recombinant IGF-I by about 12 h and was also abolished in the presence of cycloheximide or by the addition of antibodies against IGF-I. Furthermore, 17 beta E2 induced a 2- to 2.5-fold elevation of the level of IGF-I mRNA within 2-4 h, which persisted thereafter. The E2 stimulation of IGF-I mRNA was not blocked by cycloheximide, suggesting that de novo protein synthesis of an intermediate protein was not required. The IGF-I mRNA half-life, estimated by treating the cells with the RNA polymerase inhibitor 5,6-dichloro-1 beta-D-ribofuranosylbenzimidazole, was about 7 h and was not altered by E2 treatment. On the other hand, nuclear run-on assays indicated that E2 increased the transcriptional activity of the IGF-I gene, and this effect was further enhanced in cells overexpressing E2 receptors after transient transfection. These findings suggest that IGF-I may serve as a mediator for the anabolic effects of E2 on bone, and that E2 stimulates IGF-I gene expression at least in part through transcriptional control.

Animals↗
PubMed1991

Gene expression in osteoblastic cells.

Osteoblasts, the bone-forming cells, synthesize the macromolecules of the bone matrix including: type I collagen; osteocalcin; osteonectin; osteopontin; proteoglycan I and II; bone sialoprotein; matrix gla-protein; bone glycoprotein 75; several other proteins, which have not been extensively characterized; growth factors, including transforming growth factor beta and fibroblast growth factor. Osteoblasts also have high levels of the membrane-bound enzyme, alkaline phosphatase, which plays a role in matrix mineralization, and receptors for tissue-specific hormones, such as parathyroid hormone, as well as many other hormones, cytokines and growth factors, which regulate bone growth, differentiation and metabolism. The expression of these various proteins, most of which are not unique to bone but which together characterize the bone phenotype, is induced during osteoblastic differentiation in a stepwise fashion, suggestive of multiple regulatory factors. The detailed sequence of the expression of osteoblastic genes in situ has not been fully characterized. It appears that type I collagen and alkaline phosphatase are expressed early during the commitment to the osteoblastic phenotype, whereas osteopontin and osteocalcin appear late during osteoblastic differentiation. Diversity among "osteoblastic" cells is also apparent, probably not all osteoblastic cells express all the features. A large number of osteoblastic models are currently available to study the expression of osteoblast-related genes in vitro. These include primary cultures from calvaria or trabecular bone from several species, including humans, osteosarcoma-derived cell lines, and experimentally immortalized cells. Some of these in vitro models, especially the calvaria-derived cultures, undergo changes which mimic osteoblastic differentiation in vivo. The study of these and other cell models started providing insights into the regulation of gene expression in osteoblastic cells. In addition to a vast body of information on the conditions required for the expression of various proteins in culture and their regulation by hormones and growth factors, more detailed information on specific genes has recently been obtained. For example, regulation of type I collagen gene expression has been studied in osteosarcoma cell lines where 1,25(OH)2 vitamin D3 was shown to act via specific DNA segment(s) in the 5' flanking region of the gene, while parathyroid hormone affected gene expression by altering the stability of the transcripts. TGF beta 1, which stimulates osteogenesis, was shown to promote the transcription of osteopontin and type I collagen, the latter effect requiring the binding site for the transactivating protein, nuclear factor I.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

IL-1 binds to high affinity receptors on human osteosarcoma cells and potentiates prostaglandin E2 stimulation of cAMP production.

IL-1 is a potent bone resorbing agent. Its mechanism of action is unknown, but the presence of osteoblasts was shown to be necessary for IL-1 stimulation of bone resorption by isolated osteoclasts. This study examines the presence of IL-1R and IL-1 effects in osteoblastic cells from a clonal human osteosarcoma cell line, Saos-2/B-10. We found that the binding affinity and the number of binding sites increases substantially during the postconfluent stage. Scatchard and curve-fitting analysis revealed one class of high affinity binding sites, with Kd/Ki's of 40 +/- 17 pM (mean +/- SD) for IL-1 alpha (n = 5) and 9 +/- 7 pM for IL-1 beta (n = 5) and 2916 +/- 2438 (n = 6) receptors/cell. Incubation of the cells with 125I-IL-1 alpha (100 pM) at 4 degrees C, followed by incubation at 37 degrees C up to 4 h, revealed internalization of receptor-bound IL-1 alpha. Chemical cross-linking studies showed that the IL-1R in Saos-2/B-10 cells had a molecular mass of approximately 80 kDa. To assess the biologic effect of IL-1 in Saos-2/B-10 cells, we determined PGE2 content and adenylate cyclase activity. Although IL-1 had no effect on PGE2 synthesis, both IL-1 alpha and IL-1 beta enhanced PGE2 stimulation of adenylate cyclase two- to four-fold in a dose-dependent manner. The half-maximal effect for IL-1 alpha was seen at 8 to 10 pM and for IL-1 beta at 0.6 to 1.8 pM. IL-1 did not enhance basal adenylate cyclase or stimulation by parathyroid hormone, isoproterenol, or forskolin. IL-1 enhancement of PGE2-stimulated adenylate cyclase was detected between 1 to 2 h, was maximal at 4 to 5 h, was not prevented by cycloheximide treatment, and was seen in membranes from IL-1 pretreated cells. These data show effects of IL-1 on a human osteoblast-like cell line that are mediated by high affinity receptors. These IL-1 effects could contribute to the biologic action of IL-1 on bone.

Adenylyl Cyclases↗
PubMed1990

Aminohydroxybutane bisphosphonate inhibits bone loss due to immobilization in rats.

The purpose of this study was to document the effects of aminobutane bisphosphonate (AHBuP) on bone remodeling during immobilization in rats. Male Sprague-Dawley rats underwent unilateral sciatic neurectomy after receiving two daily subcutaneous injections of 0, 0.01, 0.10, or 1.0 mg P per kg AHBuP. Rats were sacrificed at 24 h or 10 or 20 days postimmobilization. Femora were ashed and tibiae were prepared for histomorphometric analysis. AHBuP was effective in inhibiting bone loss due to immobilization in a dose-dependent manner. The percentage loss of femoral ash weight due to immobilization decreased in a dose-dependent manner. In vehicle-treated rats, there was a significant decrease in trabecular bone volume (TBV) in the immobilized tibiae compared to the normal tibiae; in AHBuP-treated rats there was a dose-dependent increase in TBV both in the immobilized and control tibiae. The osteoid surface extent was decreased in AHBuP-treated rats in a dose-dependent manner. The mineral apposition rate was altered only in the intact leg of rats treated with 0.1 and 1.0 mg P AHBuP per kg compared to vehicle treated. Osteoclast number per mm was reduced by AHBuP treatment. In conclusion, aminohydroxybutane bisphosphonate effectively prevented the bone loss due to immobilization in this system.

Alendronate↗
PubMed1990

Different pattern of alkaline phosphatase, osteopontin, and osteocalcin expression in developing rat bone visualized by in situ hybridization.

Alkaline phosphatase (AP), osteopontin (OP), and osteocalcin (OC) are expressed during osteoblastic differentiation. However, previous studies suggested differences in the timing and possibly the site of their expression. In this study we used in situ hybridization to follow the distribution of these osteoblastic markers during bone development. Frozen sections of neonatal rat long bones and calvariae were hybridized with 35S-labeled RNA probes complementary to the AP, OP, and OC mRNAs. Controls included sections hybridized with the sense (mRNA) probes or pretreated with RNase. Positive cells were identified in all areas of bone formation of the long bones and calvariae. Based on quantitative silver grain distribution and density, high levels of OP expression were present only in osteoblasts in close proximity to bone (one to two cell layers). OC expression, apparently at lower levels than OP, was also localized to osteoblasts in contact with bone. In contrast AP, which was expressed at lower levels than OP, was present in a large number of cells, including preosteoblasts that were many layers removed from the bone-forming surface. These findings are consistent with the asynchronous expression of phenotypically related genes and suggest that AP is an earlier differentiation marker than OP and OC during the formation of endochondral and membranous bone.

Alkaline Phosphatase↗
PubMed1990

A model for investigating the local action of bone-acting agents in vivo: effects of hPTH(1-34) on the secondary spongiosa in the rat.

Cytokines and other local factors are likely to play an important role in bone remodeling. The purpose of this study was to develop an experimental model for evaluating the local action of various hormones and other agents on rat femur trabecular bone in vivo. Through a 1 mm diameter hole (1 mm deep) on the lateral aspect of the distal cortex of a rat femur (0.9 cm above the joint), a polyethylene tube was inserted and glued onto the bone. This tube was connected to a vascular-access-port which was implanted subcutaneously in the hip area of male Sprague-Dawley rats weighing 190-210 g. To evaluate this model, a single 50 microliters injection of 10(-8), 10(-10), or 10(-12) M hPTH(1-34) was given 1 day after catheter implantation and the number of osteoclasts was estimated 30 hours later. Bones were fixed, embedded, and stained with Masson's Trichrome stain and subjected to histomorphometric analysis. The single local parathyroid hormone (PTH) injection caused a dose-dependent increase in osteoclast number from 1.7 +/- 0.3 for phosphate-buffered saline (PBS) controls to 3.4 +/- 0.5, 5.8 +/- 0.9, and 5.4 +/- 1.0/mm for 10(-12), 10(-10), and 10(-8) M PTH, respectively. There was no increase in osteoclast number in the femurs of PBS-injected control rats, in the femurs of sham-operated rats, or in the untreated contralateral femur of the PTH-injected rat. The local injection of hPTH(1-34) did not change serum calcium, serum phosphate, or the urinary phosphate/creatinine ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Cloning and analysis of the 5' region of the rat bone/liver/kidney/placenta alkaline phosphatase gene. A dual-function promoter.

The rat bone/liver/kidney/placenta (BLKP) alkaline phosphatase (ALP) gene is expressed at high level in these particular tissues and at low levels in many other tissues. To study the mechanisms underlying the complex regulation of the rat BLKP ALP expression, we isolated a genomic clone, containing a 10.5-kb insert, which includes the promoter of the BLKP ALP gene with 2 kb of 5' flanking region, its first exon (84bp), and over 7 kb of the first intron. The promoter of the rat BLKP ALP displays features of a "housekeeping" gene promoter: an atypical TATA-box (TTCATAA); 3 potential Spl binding sites; high GC content (82% in positions-134 to -14); and a high CpG to GpC ratio (60:89 in the 0.85 kb promoter region), indicating an abundance of potential methylation sites. Likewise, transient transfection of CAT fusion genes into ROS 17/2.8 osteoblast-like cells reveals weak expression from the promoter and proximal 5' flanking sequences, which can be elevated by an SV40E enhancer. The homologous human bone/liver/kidney (BLK) ALP promoter, which demonstrates a similar combination of tissue-specific and housekeeping characteristics, shares close similarity (184 bp of 79% similarity excluding gaps) with the rat BLKP ALP promoter. The human placental ALP is encoded by a separate gene and its promoter, on the other hand lacks significant similarity to the rat BLKP ALP promoter despite their common expression in the placenta. This lack of similarity appears to reflect the close evolutionary relationship of the human placental ALP gene to the intestinal ALP gene. Significant sequence similarity was found between the rat and human BLK/BLKP ALP promoters and the human and mouse adenine deaminase promoters, and together they may represent a class of dual-function promoters, allowing both constitutive low-level, and tissue-specific higher levels of expression. A pentanucleotide with the consensus sequence 5'-GGCTC-3' is present in these promoters and in the promoters for the human fibronectin and the human alpha 1(II) procollagen genes in the region of maximal similarity with the rat BLKP ALP promoter, and in the vicinity of the Sp1-binding sites.

Alkaline Phosphatase↗
PubMed1990

Direct effects of 17 beta-estradiol on trabecular bone in ovariectomized rats.

High-affinity nuclear binding sites for 17 beta-estradiol (17 beta E2) were recently found in bone cells; however, the mechanism by which estrogen exerts its effect on bone in vivo is still unknown. To study if estrogen acts on bone directly, we used an experimental model in which test substances are infused locally into rat femur trabecular bone. Sprague-Dawley rats weighing 150-160 g were ovariectomized (OVX) and 14 days later a polyethylene tube (1 mm in diameter) connected to an Alzet osmotic minipump was implanted into the distal femur 9 mm from the joint. 17 beta E2 (24 microliters/day at 0.01-1 nM), 17 alpha-estradiol (17 alpha E2) (24 microliters/day at 1 nM), or phosphate-buffered saline (NaCl, 8 g/liter; KCl, 0.2 g/liter; KH2PO4, 0.2 g/liter; Na2HPO4.7H2O, 2.16 g/liter) was infused for 8 days. The contralateral limb remained intact. Animals were sacrificed and bones were examined by histomorphometry. Ovariectomy caused a 50% loss in trabecular bone volume (TBV) in the secondary spongiosa (from 20.3% +/- 1.7% to 9.6% +/- 1.1%; mean +/- SEM), a 2-fold increase in osteoclast number (to 4.0 +/- 0.4 per mm), a 3-fold increase in relative resorption surfaces (to 24.8% +/- 2.9%), a 9-fold increase in osteoblast number (to 11.3 +/- 2.1 per mm), and an 8-fold increase in relative osteoid surface (to 9.6% +/- 1.7%). The local infusion of 17 beta E2 for 8 days into OVX rats (i) restored the TBV dose dependently to 75% and 85% of control (non-OVX) levels, at 0.1 nM and 1 nM 17 beta E2, respectively; (ii) decreased osteoclast number and the relative resorption surface to control (non-OVX) levels; and (iii) further increased osteoblast number and the relative osteoid surface dose dependently (by 5-fold at 1 nM 17 beta E2). Phosphate-buffered saline infusion was without effect. Infusion of 17 alpha E2 had no effect on TBV, osteoclast number, or resorption surface but increased slightly the osteoblast number and the osteoid surface. Its potency was 1/100 that of 17 beta E2. The local infusion of 17 beta E2 or 17 alpha E2 had no effect on body or uterine weight. We conclude from these findings that estrogen delivered directly to the bone of OVX rats in vivo at 2.4 and 24 fmol/day acted locally to inhibit bone resorption and stimulate bone formation.

Animals↗
PubMed1990

The effect of hemopoietic growth factors on the generation of osteoclast-like cells in mouse bone marrow cultures.

Multinucleated cells containing tartrate-resistant acid phosphatase were produced in mouse bone marrow cultures in response to 1,25-dihydroxyvitamin D3. These cells resemble osteoclasts in their morphology, possess receptors for calcitonin, and resorb bone in culture. The effects of several hemopoietic regulatory proteins on the generation of these cells were examined in this study. Interleukin-3, granulocyte-macrophage-stimulating factor (GMCSF), and macrophage-stimulating factor strongly inhibited generation of the tartrate-resistant acid phosphatase-containing multinucleated cells with approximate EC50 values of 3, 6, and 3 colony-forming units/ml, respectively. Granulocyte colony stimulating factor, interleukin-6, and leukemia inhibitory factor had no effect on the generation of these cells. In addition, we observed that the number of these cells was reduced when the bone marrow was plated at high cell density, and that this inhibitory effect was reversed by the addition of neutralizing antibodies directed against GMCSF. These findings suggest that GMCSF and other hemopoietic factors secreted by cells in the bone marrow regulate development of the osteoclast-like cells, possibly by diverting common precursor cells to alternate pathways.

Acid Phosphatase↗
PubMed1990

Biphasic effects of transforming growth factor-beta on the production of osteoclast-like cells in mouse bone marrow cultures: the role of prostaglandins in the generation of these cells.

Osteoclast-like multinucleated giant cells are induced in bone marrow cultures by 1,25-dihydroxyvitamin D3 and other agents. These cells resemble osteoclasts in their morphology, their ability to resorb bone, and the possession of calcitonin receptors. We report here a biphasic effect of transforming growth factor-beta (TGF beta) on the generation of these cells in mouse bone marrow cultures. At low concentrations (10-100 pg/ml) TGF beta enhanced 1,25-dihydroxyvitamin D3-dependent production of osteoclast-like cells, while at higher concentrations TGF beta was inhibitory. Complete inhibition was seen at 4 ng/ml. Antibodies directed against TGF beta significantly reduced the generation of osteoclast-like cells in 1,25-dihydroxyvitamin D3-treated cultures, indicating the contribution of endogenous TGF beta activity. TGF beta also enhanced the accumulation of prostaglandin E2 (PGE2) in a dose-dependent manner. Moreover, we found that the generation of these cells in response to 1,25-dihydroxyvitamin D3 was also dependent on PG accumulation, since it was inhibited by indomethacin (250 ng/ml), and this inhibition could be reversed by exogenous PGE2. It is, thus, suggested that PG activity, probably PGE2, mediates the enhancing effect of low TGF beta concentrations and is required for the generation of osteoclast-like cells in this system.

Acid Phosphatase↗