Transcriptional regulation of osteopontin production in rat osteoblast-like cells by parathyroid hormone.

Osteopontin (OP) or bone sialoprotein is a recently characterized extracellular matrix protein which is abundant in bone and is produced by osteoblasts. Parathyroid hormone (PTH) is a potent calcitropic hormone which regulates osteoblastic function including the synthesis of extracellular matrix proteins. This study examines the effect of human PTH (hPTH-[1-34]) on the expression of this novel protein in rat osteoblast-like cells. hPTH(1-34) significantly decreased the amount of OP in culture media of the rat osteoblastic osteosarcoma cell line, ROS 17/2.8, detected by Western immunoblot analysis. hPTH(1-34) also suppressed the steady-state level of OP mRNA two- to threefold with an ED50 of approximately 3 X 10(-10) M. This inhibition was detectable at 24 h, reached its nadir at 48 h, and lasted at least up to 96 h. The hPTH(1-34) effects were mimicked by isobutylmethylxanthine, cholera toxin, 8-bromo-cAMP, forskolin, and isoproterenol. hPTH(1-34) suppressed by two- to threefold the rate of OP gene transcription, estimated by nuclear run-on assays. The suppression of OP mRNA levels by hPTH(1-34) was also seen when basal levels were increased by transforming growth factor type beta, or 1,25-dihydroxyvitamin D3, or were decreased by dexamethasone. A similar decrease in the steady-state level of OP mRNA by hPTH(1-34) was also observed in primary cultures of osteoblast-enriched cells from fetal rat calvaria. These findings indicate that hPTH(1-34) suppresses the production of the novel extracellular matrix protein, OP, in osteoblasts at least in part through transcriptional control.

Convergence of alpha(v)beta(3) integrin- and macrophage colony stimulating factor-mediated signals on phospholipase Cgamma in prefusion osteoclasts.

The macrophage colony stimulating factor (M-CSF) and alpha(v)beta(3) integrins play critical roles in osteoclast function. This study examines M-CSF- and adhesion-induced signaling in prefusion osteoclasts (pOCs) derived from Src-deficient and wild-type mice. Src-deficient cells attach to but do not spread on vitronectin (Vn)-coated surfaces and, contrary to wild-type cells, their adhesion does not lead to tyrosine phosphorylation of molecules activated by adhesion, including PYK2, p130(Cas), paxillin, and PLC-gamma. However, in response to M-CSF, Src(-/-) pOCs spread and migrate on Vn in an alpha(v)beta(3)-dependent manner. Involvement of PLC-gamma activation is suggested by using a PLC inhibitor, U73122, which blocks both adhesion- and M-CSF-mediated cell spreading. Furthermore, in Src(-/-) pOCs M-CSF, together with filamentous actin, causes recruitment of beta(3) integrin and PLC-gamma to adhesion contacts and induces stable association of beta(3) integrin with PLC-gamma, phosphatidylinositol 3-kinase, and PYK2. Moreover, direct interaction of PYK2 and PLC-gamma can be induced by either adhesion or M-CSF, suggesting that this interaction may enable the formation of integrin-associated complexes. Furthermore, this study suggests that in pOCs PLC-gamma is a common downstream mediator for adhesion and growth factor signals. M-CSF-initiated signaling modulates the alpha(v)beta(3) integrin-mediated cytoskeletal reorganization in prefusion osteoclasts in the absence of c-Src, possibly via PLC-gamma.

High lateral mobility of endogenous and transfected alkaline phosphatase: a phosphatidylinositol-anchored membrane protein.

The lateral mobility of alkaline phosphatase (AP) in the plasma membrane of osteoblastic and nonosteoblastic cells was estimated by fluorescence redistribution after photobleaching in embryonic and in tumor cells, in cells that express AP naturally, and in cells transfected with an expression vector containing AP cDNA. The diffusion coefficient (D) and the mobile fraction, estimated from the percent recovery (%R), were found to be cell-type dependent ranging from (0.58 +/- 0.16) X 10(-9) cm2s-1 and 73.3 +/- 10.5 in rat osteosarcoma cells ROS 17/2.8 to (1.77 +/- 0.51) X 10(-9) cm2s-1 and 82.8 +/- 2.5 in rat osteosarcoma cells UMR106. Similar values of D greater than or equal to 10(-9) cm2s-1 with approximately 80% recovery were also found in fetal rat calvaria cells, transfected skin fibroblasts, and transfected AP-negative osteosarcoma cells ROS 25/1. These values of D are many times greater than "typical" values for membrane proteins, coming close to those of membrane lipid in fetal rat calvaria and ROS 17/2.8 cells (D = [4(-5)] X 10(-9) cm2s-1 with 75-80% recovery), estimated with the hexadecanoyl aminofluorescein probe. In all cell types, phosphatidylinositol (PI)-specific phospholipase C released 60-90% of native and transfection-expressed AP, demonstrating that, as in other tissue types, AP in these cells is anchored in the membrane via a linkage to PI. These results indicate that the transfected cells used in this study possess the machinery for AP insertion into the membrane and its binding to PI. The fast AP mobility appears to be an intrinsic property of the way the protein is anchored in the membrane, a conclusion with general implications for the understanding of the slow diffusion of other membrane proteins.

Membrane changes during cartilage maturation. Increase in 5'-nucleotidase and decrease in adenosine inhibition of adenylate cyclase.

To examine the potential participation of the plasma membrane in differentiation, we studied the enzymatic activities of 5'-nucleotidase and adenylate cyclase as a function of chondrocyte maturation. 16-day-old chick embryo tibiae epiphyses were dissected into proliferative, growing and hypertrophying zones. Partially purified membrane fractions prepared by differential centrifugation from the respective tissue segments were assayed for enzymatic activity. Cell suspensions from the same segments were examined cytochemically for the presence of 5'-nucleotidase. The findings show that the 5'-nucleotidase activity of the chick embryo epiphyseal cartilage has the following characteristics: (a) it has a Km of about 25 muM for 5'AMP, and is inhibited by a mixture of 2' and 3'AMP (apparent Ki about 10(-4) M) and by AOPCP; (b) it is predominantly localized at the cell surface but is also detected in the cytoplasm and in association with nuclear heterochromatin; and (c) it increases 10-fold (on a DNA basis) during the maturation of the epiphyseal cartilage cells. The adenylate cyclase activity has these characteristics: (a) it does not change during chondrocyte maturation (on a DNA basis); (b) its susceptibility to adenosine inhibition decreases at least 10-fold. The implication of these findings relative to a possible role of adenosine in cellular communication is discussed.

Therapeutic approaches to bone diseases.

The strength and integrity of our bones depends on maintaining a delicate balance between bone resorption by osteoclasts and bone formation by osteoblasts. As we age or as a result of disease, this delicate balancing act becomes tipped in favor of osteoclasts so that bone resorption exceeds bone formation, rendering bones brittle and prone to fracture. A better understanding of the biology of osteoclasts and osteoblasts is providing opportunities for developing therapeutics to treat diseases of bone. Drugs that inhibit the formation or activity of osteoclasts are valuable for treating osteoporosis, Paget's disease, and inflammation of bone associated with rheumatoid arthritis or periodontal disease. Far less attention has been paid to promoting bone formation with, for example, growth factors or hormones, an approach that would be a valuable adjunct therapy for patients receiving inhibitors of bone resorption.

Expression of a calcium-mobilizing parathyroid hormone-like peptide in lactating mammary tissue.

A survey of rat tissues by RNA analysis, aimed at uncovering the physiological function of the parathyroid hormone-like peptide (PTH-LP) associated with hypercalcemia of malignancy, revealed the presence of a 1.5-kilobase messenger RNA encoding this peptide in lactating mammary glands. PTH-LP messenger RNA is expressed in mammary tissue only during lactation; it appears and disappears rapidly (2 to 4 hours) as a function of the sucking stimulus. The identity of this messenger RNA was confirmed by cloning the rat PTH-LP complementary DNA, which predicts a peptide with strong similarity to the human homolog. Moreover, extracts from lactating mammary tissue stimulated parathyroid hormone-dependent adenylate cyclase. These findings suggest that PTH-LP plays a physiological role in lactation, possibly as a hormone for the mobilization or transfer (or both) of calcium to the milk.

Cyclic AMP and cyclic GMP: mediators of the mechanical effects on bone remodeling.

Compressive forces of physiological magnitude (60 grams per square centimeter) reduce the adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate content of the epiphyses of tibiae from 16-day-old chick embryos. An equivalent hydrostatic pressure applied directly to cells isolated from this tissue also affects cyclic nucleotide accumulation. The tissue response is uniform throughout the epiphysis, whereas the cell response varies according to the area of origin.

DNA synthesis in cartilage cells is stimulated by oscillating electric fields.

External oscillating electric fields (1166 volts per centimeter, 5 hertz) enhanced the incorporation of [3H] thymidine into the DNA of chondrocytes isolated from the proliferative layer of embryonic (16 days) chick epiphysis. Verapamil or tetrodotoxin at 10(-6)M concentrations completely blocked the electric field effect. Tetracaine reduced the incorporation of [3H] thymidine in both control and electrically stimulated cells. The findings support the hypothesis that Na and Ca2 fluxes generated by the electrical perturbation trigger DNA synthesis in these cells.

Acid extrusion is induced by osteoclast attachment to bone. Inhibition by alendronate and calcitonin.

Acid extrusion is essential for osteoclast (OC) activity. We examined Na+ and HCO3(-)-independent H+ extrusion in rat- and mouse OCs by measuring intracellular pH (pHi) changes, with the pHi indicator BCECF (biscarboxyethyl-5-(6) carboxyfluorescein) after H+ loading with an ammonium pulse. 90% of OCs attached to glass do not possess HCO3- and Na(+)-independent H(+)-extrusion (rate of pHi recovery = 0.043 +/- 0.007 (SEM) pH U/min, n = 26). In contrast, in OCs attached to bone, the pHi recovery rate is 0.228 +/- 0.011 pHi U/min, n = 25. OCs on bone also possess a NH(4+)-permeable pathway not seen on glass. The bone-induced H+ extrusion was inhibited by salmon calcitonin (10(-8) M, for 2 h), and was not present after pretreating the bone slices with the aminobisphosphonate alendronate (ALN). At ALN levels of 0.22 nmol/mm2 bone, H+ extrusion was virtually absent 12 h after cell seeding (0.004 +/- 0.002 pH U/min) and approximately 50% inhibition was observed at 0.022 pmol ALN/mm2 bone. The Na(+)-independent H+ extrusion was not inhibited by bafilomycin A1 (up to 10(-7) M), although a bafilomycin A1 (10(-8) M)-sensitive H+ pump was present in membrane vesicles isolated from these osteoclasts. These findings indicate that Na(+)-independent acid extrusion is stimulated by osteoclast attachment to bone and is virtually absent when bone is preincubated with ALN, or when osteoclasts are treated with salmon calcitonin.

Comparison of postreceptor effects of 1-34 human hypercalcemia factor and 1-34 human parathyroid hormone in rat osteosarcoma cells.

A tumor-derived factor believed to cause hypercalcemia by acting on the parathyroid hormone (PTH) receptor was recently purified, cloned, and found to have NH2-terminal sequence homology with PTH. The 1-34 region of this protein was synthesized, evaluated for its postreceptor effects on the ROS 17/2.8 cell line, and its properties were compared to 1-34 PTH. Both 1-34 human humoral hypercalcemia factor (HCF) and 1-34 PTH stimulated adenylate cyclase with an effective concentration (EC)50 of approximately 1 nM. The extent of stimulation by both peptides was equally enhanced by dexamethasone. They both had a pronounced inhibitory effect on growth in the presence of dexamethasone, with an EC50 of approximately 0.1 nM, reduced alkaline phosphatase (AP) activity by approximately 70% in the absence of dexamethasone and by approximately 80% in the presence of dexamethasone with an EC50 of 0.03 nM, and when present at a concentration of 10 nM, reduced AP mRNA levels (estimated by Northern analysis) by approximately 80% in the presence or absence of dexamethasone. Thus, in addition to similar dose-response curves for adenylate cyclase stimulation, both HCF and PTH produced identical postreceptor effects in ROS 17/2.8 cells. These effects of HCF are probably mediated by the interaction of the tumor-derived factor with the PTH receptor.

PYK2 in osteoclasts is an adhesion kinase, localized in the sealing zone, activated by ligation of alpha(v)beta3 integrin, and phosphorylated by src kinase.

Osteoclast activation is initiated by adhesion to the bone surface, followed by cytoskeletal rearrangement, the formation of the sealing zone, and a polarized ruffled membrane. This study shows that PYK2/CAKbeta/RAFTK, a cytoplasmic kinase related to the focal adhesion kinase, is highly expressed in rat osteoclasts in vivo. Using murine osteoclast-like cells (OCLs) or their mononuclear precursors (pOCs), generated in a coculture of bone marrow and osteoblastic MB1.8 cells, we show: (a) tyrosine phosphorylation of PYK2 upon ligation of beta3 integrins or adhesion of pOCs to serum, vitronectin, osteopontin, or fibronectin but not to laminin or collagen; (b) coimmunoprecipitation of PYK2 and c-Src from OCLs; (c) PYK2 binding to the SH2 domains of Src; (d) marked reduction in tyrosine phosphorylation and kinase activity of PYK2 in OCLs derived from Src (-/-) mice, which do not form actin rings and do not resorb bone; (e) PYK2 phosphorylation by exogeneous c-Src; (f) translocation of PYK2 to the Triton X-100 insoluble cytoskeletal fraction upon adhesion; (g) localization of PYK2 in podosomes and the ring-like structures in OCLs plated on glass and in the sealing zone in OCLs plated on bone; and (h) activation of PYK2, in the presence of MB1.8 cells, parallels the formation of sealing zones and pit resorption in vitro and is reduced by echistatin or calcitonin and cytochalasin D. Taken together, these findings suggest that Src-dependent tyrosine phosphorylation of PYK2 is involved in the adhesion-induced formation of the sealing zone, required for osteoclastic bone resorption.

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.

Induction of vascular endothelial growth factor expression by prostaglandin E2 and E1 in osteoblasts.

PGE1 and PGE2 are potent stimulators of bone formation. Osteogenesis is strongly dependent on angiogenesis. Vascular endothelial growth factor (VEFG), a secreted endothelial cell-specific mitogen, has been implicated in physiological and pathological angiogenesis. The aim of this study was to examine the possible role of VEGF in PG stimulation of bone formation. We found that in rat calvaria-derived osteoblast-enriched cells and in the osteoblastic RCT-3 cell line PGE2 and E1 increased VEGF mRNA and protein levels. The increased expression of VEGF mRNA produced by PGE2 was rapid (maximal at 1 h), transient (declined by 3 h), potentiated by cycloheximide, and abolished by actinomycin D. PGE2 had no effect on VEGF mRNA stability, suggesting transcriptional regulation of VEGF expression by PGF2. Rp-cAMP, a cAMP antagonist, suppressed VEGF mRNA induced by PGE2, indicating cAMP mediation. The upregulation of VEGF expression by PGE2 in the preosteoblastic RCT-1 cells was potentiated by treatment with retinoic acid, which induces the differentiation of these cells. The upregulation of VEGF mRNA by PGE2 was inhibited by dexamethasone treatment. In addition, Northern blot analysis showed that VEGF mRNA is expressed in adult rat tibia. In summary, we documented, for the first time, the expression of VEGF in osteoblasts and in bone tissue. Stimulation of VEGF expression by PGs and its suppression by glucocorticoids, which, respectively, stimulate and suppress bone formation, strongly implicate the involvement of VEGF in bone metabolism.

Comparison of alendronate and sodium fluoride effects on cancellous and cortical bone in minipigs. A one-year study.

Fluoride stimulates trabecular bone formation, whereas bisphosphonates reduce bone resorption and turnover. Fracture prevention has not been convincingly demonstrated for either treatment so far. We compared the effects of 1-yr treatment of 9-mo-old minipigs with sodium fluoride (NaF, 2 mg/kg/d p.o.) or alendronate (ALN, 4 amino-1-hydroxybutylidene bisphosphonate monosodium, 1 mg/kg/d p.o.) on the biomechanical and histomorphometric properties of pig bones. As expected, NaF increased and ALN decreased bone turnover, but in these normal animals neither changed mean bone volume. NaF reduced the strength of cancellous bone from the L4 vertebra, relative to control animals, and the stiffness (resistance to deformation) of the femora, relative to the ALN group. In the ALN-treated animals, there was a strong positive correlation between bone strength and L5 cancellous bone volume, but no such correlation was observed in the NaF group. Furthermore, the modulus (resistance to deformation of the tissue) was inversely related to NaF content and there was a relative decrease in bone strength above 0.25 mg NaF/g bone. Moreover, within the range of changes measured in this study, there was an inverse correlation between bone turnover, estimated as the percentage of osteoid surface, and modulus. These findings have relevant implications regarding the use of these agents for osteoporosis therapy.

Factors associated with humoral hypercalcemia of malignancy stimulate adenylate cyclase in osteoblastic cells.

The culture media of three cell lines, a human prostate carcinoma (PC3), a rat Leydig cell tumor (Rice-500), and a rat carcinosarcoma (WRC-256), that were derived from tumors associated with humoral hypercalcemia of malignancy (HHM), were examined for stimulation of adenylate cyclase in ROS 17/2.8 osteoblastic cells and for bone resorptive activity in culture. Cells from a nonhypercalcemic variant of the WRC256 tumor served as control. Extracts from three solid human tumors, a lung adenocarcinoma from a patient with HHM and two adenocarcinoma from normocalcemic patients (lung and colon), were also examined for adenylate cyclase stimulation. We found excellent correlation between stimulation of cyclic AMP accumulation in ROS 17/2.8 cells and bone resorbing activity in culture, or production of HHM in vivo. Stimulation of adenylate cyclase by HHM factors was inhibited by the parathyroid hormone competitive inhibitor, [8norleucyl, 18norleucyl, 34tyrosinyl] bovine parathyroid hormone (3-34) amide.

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.

Preservation of thoracic spine microarchitecture by alendronate: comparison of histology and microCT.

The effect of bisphosphonates on trabecular microarchitecture may contribute to the reduced risk of vertebral fracture with treatment independent of the bone volume. Trabecular structure was examined at the twelfth thoracic vertebra after 2 years of treatment of two groups of ovariectomized baboons on high and low doses of alendronate, compared with ovariectomized and non-ovariectomized controls. Standard 2D histological measurements showed that alendronate treatment of ovariectomized animals resulted in significantly higher total trabecular length and a lower marrow star volume in comparison with ovariectomized controls indicating preservation of connectivity. Similarly when the vertebrae were examined using a novel thick slice technique that combines 2D and 3D information, ovariectomy produced a significantly higher number of "real" trabecular termini in comparison with normal. When ovariectomized animals were treated with increasing doses of alendronate, fewer "real" termini were seen. MicroCT analysis (2D and 3D) correlated well with the histological measurements, although more variability and less discrimination between groups was seen, with no statistically significant differences with alendronate treatment. Reduced vertebral fracture risk with alendronate may be due to a combination of factors including the increased bone volume, reduced turnover and greater mineralization reported by others. Added to this is now suggested the preservation of several aspects of vertebral cancellous architecture, with microscopy the most sensitive method of analysis.

Prostaglandin E2 (PGE2) increases the number of rat bone marrow osteogenic stromal cells (BMSC) via binding the EP4 receptor, activating sphingosine kinase and inhibiting caspase activity.

Prostaglandin E(2) (PGE(2)) is bone-anabolic, i.e. stimulates bone formation and increases bone mass. In this study, we explored possible intracellular mechanisms of its increase of osteogenic cells in rat bone marrow. Adherent rat bone marrow cells were counted after 12-48 h or cultured for 21 days and mineralized nodules were counted. Also, apoptosis of marrow cells was measured after in vivo PGE(2) injection. PGE(2) (100 nM) increased 2-3 fold the number of adherent BMSC, an effect which was mediated via binding the EP(4) receptor since it was mimicked by forskolin and 11-deoxy-prostaglandin E(1) (PGE(1)) and was blocked by DDA and L-161982 (EP(4) antagonist). PGE(2) stimulated sphingosine kinase (SPK) activity since its effects were blocked by DMS (SPK inhibitor) and mimicked by SPP (SPK product). PGE(2) reduced the activity of caspase-3 and -8 in BMSC and their inhibitors increased BMSC number and nodule formation. In vivo, PGE(2) prevented the increase in the apoptosis of bone marrow cells caused by indomethacin. We propose that PGE(2) exerts an anti-apoptotic effect on BMSC, thereby increasing their number and subsequent osteoblastic differentiation. Such an effect could explain how PGE(2) stimulates bone formation in vivo.

Characterization of a human osteosarcoma cell line (Saos-2) with osteoblastic properties.

This study examines the osteoblastic properties of the established human osteosarcoma cell line Saos-2. Saos-2 cells inoculated into diffusion chambers, which were implanted i.p. into nude mice, produced mineralized matrix in 4 of 6 chambers at 8 weeks. In 5 of 6 chambers there was a strong positive alkaline phosphatase reaction. In culture the alkaline phosphatase levels increased with time and cell density, reaching very high levels at confluence: 4-7 mumol/mg protein/min. The cells show a sensitive adenylate cyclase response to parathyroid hormone, 50% effective dose = 2.8 nM, which increases with cell density and is further raised by dexamethasone treatment. They also exhibit typical binding of 1-25-dihydroxyvitamin D3 to 3.2S receptor protein with an apparent Kd of 0.21 nM; the numbers of sites per cell were 3,300 at 50,000 cells/cm2 and 1,800 at 280,000 cells/cm2. The presence of osteonectin was visualized with a monoclonal antibody which revealed a reticular pattern on the cell surface. Osteonectin was also detected in the medium by Western blots, migrating at around Mr 40,000 in nonreduced gels and Mr 44,000 in reduced gels. The Saos-2 cells thus possess several osteoblastic features and could be useful as a permanent line of human osteoblast-like cells and as a source of bone-related molecules.

Microtubule disruption enhances prostaglandin E2 production in osteoblastic cells.

Prostaglandins have been implicated in the response of bone to mechanical stimuli. To explore the potential role of the cytoskeleton in the control of prostaglandin production, we examined the effect of cytoskeleton disrupting agents on arachidonic acid metabolism in rat calvaria osteoblastic cells. We found that microtubule disrupting agents increase prostaglandin E production 4-5-fold. Stimulation was first detectable at 4 h and rose sharply between 4 and 8 h. 2 h exposure to 1 microM colchicine was sufficient to produce the maximum effect. Cytochalasin B at concentrations which caused marked shape changes had no effect on prostaglandin E production or on its stimulation by colchicine. Taxol, a stabilizer of microtubules, reduced the colchicine effect. The increase in prostaglandin E production was associated with enhanced conversion of arachidonic acid to prostaglandin E2 rather than enhanced release of arachidonic acid from phospholipids. This increase in enzymatic activity was not abolished by cycloheximide treatment at concentrations which inhibited 90% of protein synthesis in the cells.