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

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

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.     

Comparison of the effects of synthetic human parathyroid hormone (PTH)-(1-34)-related peptide of malignancy and bovine PTH-(1-34) on bone formation and resorption in organ culture

We have compared the effects of synthetic amino-terminal human PTH-(1-34)-related peptide (PTHrP) of malignancy with those of synthetic bovine PTH-(1-34) in cultures of half-calvariae from 21-day-old fetal rats and of parietal bones from 7-day neonatal mice. Incorporation of [3H] proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP), and percent collagen synthesis (PCS) were measured in both systems. Incorporation of [3H]thymidine and cAMP production were measured in fetal rat calvariae. Production of prostaglandin E2 and I2 and bone resorption, as assessed by release of previously incorporated 45Ca, were measured in mouse parietal bones. The effects of PTHrP and PTH were qualitatively similar. At 96 h CDP in rat calvariae was decreased by PTH at a concentration as low as 0.01 nM, while similar effects were seen with PTHrP at 0.1 nM. Effects on NCP were small, so PCS was reduced. At 24 h [3H]thymidine was not altered, but CDP and PCS were decreased by both PTH and PTHrP. cAMP production was increased in fetal rat calvariae at 30 min. Both PTH and PTHrP increased 45Ca release at low concentrations and prostaglandin production at high concentrations in mouse parietal bones. While PTH was about 10-fold more potent than PTHrP, there was no qualitative difference in the responses. These studies further suggest that PTHrP affects bone through the PTH receptor.     

Cortisol modulates the actions of interleukin-1 alpha on bone formation, resorption, and prostaglandin production in cultured mouse parietal bones

Previous studies have shown that both interleukin-1 (IL-1) and glucocorticoids inhibit collagen synthesis in bone organ and cell cultures. In this study we examined their interactions in cultured neonatal mouse parietal bones. IL-1 alpha stimulated [3H]thymidine incorporation. Cortisol decreased thymidine incorporation, but did not block the effect of IL-1. Both cortisol and IL-1 alpha decreased the incorporation of [3H]proline into collagenase-digestible protein (CDP) and reduced alpha 1(I)procollagen mRNA levels at 72 h. Noncollagen protein (NCP) labeling was increased by IL-1 and decreased by cortisol. In the presence of cortisol, IL-1 alpha (6 pM) increased CDP as well as NCP labeling. The increase in CDP labeling was paralleled by an increase in alpha 1(I)procollagen mRNA, suggesting a pretranslational site for the cortisol-IL-1 alpha interaction. In the same bones, cortisol consistently blocked IL-1 alpha-stimulated 45Ca release and prostaglandin E2 (PGE2) production. The ability of IL-1 alpha to increase CDP in the presence of cortisol was the same in the presence or absence of indomethacin, an inhibitor of PGE2 synthesis, or aphidicholin (30 microM), an inhibitor of DNA synthesis, indicating that the reversal was neither PG mediated nor dependent on cell proliferation. In conclusion, our results demonstrate that IL-1 inhibits collagen, but not NCP or DNA, synthesis and that cortisol inhibits IL-1 alpha-induced bone resorption and PGE2 production and reverses its inhibitory effect on collagen synthesis in cultured neonatal mouse calvariae.     

Effect of sodium vanadate on deoxyribonucleic acid and protein syntheses in cultured rat calvariae

Sodium vanadate, an agent known to have multiple cellular actions, was studied for its effects on aspects of bone formation in cultures of 21-day-old fetal rat calvariae. Vanadate (0.1-10 microM) stimulated the incorporation of [3H] thymidine into acid-insoluble residues (DNA); the effect appeared after 3 h and was sustained for 96 h. Vanadate increased the bone DNA content and mitotic index. Treatment with vanadate at 10 microM for 24 h or at 0.3-1 microM for 96 h increased the incorporation of [3H]proline into collagenase-digestible protein (CDP), but the effect was not specific for collagen; vanadate also increased the labeling of noncollagen protein (NCP). Vanadate increased the incorporation of [3H]proline into type I collagen without affecting other collagen types. Vanadate (100 microM) caused a marked and irreversible inhibitory effect on the labeling of DNA, CDP, and NCP. Treatment with vanadate at multiple doses for 3-96 h did not stimulate alkaline phosphatase activity, but this enzyme was inhibited in bones exposed to 1 mM vanadate for 24 h or 10 microM vanadate for 96 h. The stimulatory effect on DNA labeling was primarily observed in the periosteum, while that on CDP labeling was seen only in the periosteum-free bone. These studies indicate that sodium vanadate stimulates bone DNA, collagen, and NCP syntheses in vitro, although high doses of vanadate have an irreversible inhibitory effect.     

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.     

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.     

Effects of the calcium antagonist verapamil on in vitro synthesis of skeletal collagen and noncollagen protein.

Effects of the calcium antagonist verapamil on the synthesis of fetal rat bone collagen and noncollagen protein were investigated in tissue culture. Protein synthesis was quantitated by measuring the incorporation of [3H]proline into collagenase-digestible (CDP) and noncollagen protein (NCP) using bacterial collagenase; [3H]proline was added for the last 2 h of culture. Verapamil (10(-5)--10(-4) M) decreased the incorporation of label into CDP and NCP after 24 h of culture; CDP formation was inhibited to a greater extent than NCP. The inhibitory response was observed in the presence and absence of unlabeled medium proline and was not associated with changes in trichloroacetic acid-extractable radioactivity. Increasing medium calcium from 1.0 to 5.0 mM did not affect the response to 10(-4) M verapamil, whereas 3.0 mM calcium abolished the response to 10(-5) M verapamil. The inhibitory effect was reversed by 48 h of control treatment subsequent to 24-h treatment with the antagonist. Verapamil did not decrease the incorporation of [3H]thymidine into DNA or [3H]uridine into RNA, nor was there any effect of the antagonist on the DNA content of cultured bones. The prostaglandin synthetase inhibitor indomethacin did not affect the response to verapamil. We conclude that a critical concentration of intracellular calcium is necessary for normal synthesis of skeletal protein in tissue culture, and that collagen may be more sensitive to changes in intracellular calcium than NCP. In addition, other ions (e.g. sodium and potassium) may also be involved in the control of skeletal protein synthesis.     

Hormonal control of bone collagen synthesis in vitro. Effects of insulin and glucagon.

The direct effects of porcine insulin and glucagon on bone collagen and non-collagen protein synthesis have been examined in cultures of calvaria obtained from 21-day fetal rats. Bones were incubated for 24 to 96 h and [3H]proline was added for the last 2 h of culture. Incorporation of the label into collagenase-digestible protein (CDP) and noncollagen protein (NCP) was determined using purified bacterial collagenase. Insulin increased the labeling of CDP by 60 to 115% at concentrations of 10(-9) to 10(-6) M. A smaller stimulatory effect was observed on NCP. The effect on CDP appeared after 12 to 24 h of culture, was maintained for 96 h in the continuous presence of the hormone, but was lost within 3 h of removal of insulin from the culture medium. Insulin appeared to have a direct effect on collagen synthesis and not on collagen breakdown. Insulin did not affect the incorporation of [3H]uridine or [3H]thymidine into the RNA and DNA fractions of bone at 24 h. Insulin opposed the inhibitory effects of parathyroid hormone and dibutyryl cyclic-3',5'-adenosine monophosphate and to a lesser extent, the inhibitory effect of isobutylmethylxanthine on the labeling of CDP. Glucagon did not affect the response to insulin and by itself had small and variable inhibitory effects on proline incorporation.     

Mechanism of action of estrogen on intramembranous bone formation: regulation of osteoblast differentiation and activity.

Dynamic bone histomorphometry, [3H]thymidine radioautography, and Northern analysis for bone matrix proteins and insulin-like growth factor-I (IGF-I) were performed in calvariae of ovariectomized (OVX) and estrogen-treated OVX rats. Treatment of OVX rats with diethylstilbestrol (DES) for 2 weeks reduced the periosteal mineral apposition rate, osteoblast number, and osteoblast size in calvarial periosteum. DES treatment also reduced the number of preosteoblasts in the S phase of the cell cycle, suggesting that the decrease in osteoblast number was due in part to inhibition of proliferation of osteoprogenitor cells. One week after ovariectomy, there were small increases in mRNA levels for pre pro-alpha 2 (I) subunit of type I collagen (collagen), osteocalcin, and osteonectin and a large increase in the mRNA level for IGF-I. DES treatment resulted in rapid decreases (3 h) in the mRNA levels for osteonectin, osteocalcin, and IGF-I. In contrast, mRNA levels for collagen were virtually unchanged after short term DES treatment. Uterus and liver served as positive and negative control tissues, respectively, for the effects of DES on IGF-I mRNA levels in OVX rats; mRNA levels were increased in uterus and decreased in liver after hormone treatment. We conclude from these studies that estrogen reduces periosteal bone formation by inhibiting both the differentiation and activity of osteoblasts. Furthermore, down-regulation of mRNA levels for IGF-I and bone matrix proteins precedes the changes in dynamic bone histomorphometry.     

Normal somatomedin-C/insulin-like growth factor I binding and action in cultured human fibroblasts from Turner syndrome

Growth retardation is a major manifestation of Turner syndrome (TS). Since plasma growth hormone and somatomedin-C/insulin-like growth factor I (SM-C/IGF-I) levels are generally normal, growth failure has been ascribed to peripheral defects in SM-C/IGF-I receptors or action. We have measured the binding of [125I]SM-C/IGF-I to cultured fibroblast monolayers derived from patients with Turner syndrome, and have evaluated SM-C/IGF-I stimulation of both [3H]thymidine incorporation and cell replication. When compared to fibroblasts from normal adults, newborns, and age-matched children, no significant differences were observed in specific binding of [125I]SM-C/IGF-I to fibroblast monolayers, and displacement curves demonstrated similar receptor affinities for all groups. Similarly, equivalent SM-C/IGF-I stimulation of [3H]thymidine incorporation was seen in both Turner and control fibroblasts. In the absence of serum, SM-C/IGF-I, at a concentration of 10-25 ng/ml, stimulated thymidine incorporation 3.7-11.8-fold in Turner fibroblasts and 1.9-9.8-fold in control cells. In combination with 1.0% human hypopituitary serum (HHS), SM-C/IGF-I stimulated thymidine incorporation 20-70-fold in all cell lines. Cell replication in both TS and control cells was increased 90% by the combination of SM-C/IGF-I + 0.5% HHS, and 140% by SM-C/IGF-I + 0.5% HHS + dexamethasone. We conclude that TS fibroblasts have normal SM-C/IGF-I receptors and sensitivity, and are capable of enhanced DNA synthesis and replication following SM-C/IGF-I stimulation.     

Parathyroid hormone potentiates the effect of insulin-like growth factor-I on bone formation

Insulin-like growth factor-I and parathyroid hormone are both known regulators of bone formation. In this study, human recombinant IGF-I and bovine PTH (1-34) and their combination were studied for their effects in vitro on the proliferation of embryonic chick osteoblast-like cells (osteoblasts) and in vivo on bone formation in normal rats. Osteoblasts from 17-day-old chick embryos were cultured in serum-free BGJb medium containing 0.1% bovine albumin. After 2 days, IGF-I and/or PTH were added. Twenty-four hours later [3H]thymidine incorporation into trichloroacetic acid precipitable material was quantified as an index of cell proliferation. This has previously been shown to reflect actual cell division. IGF-I at doses ranging from 0.85 to 13.6 nmol/l caused a dose-dependent increase in [3H]thymidine incorporation into osteoblasts. PTH alone (10 to 1000 pmol/l) had no significant effect. However, when combined with IGF-I, PTH potentiated the mitogenic effect of IGF-I and achieved statistical significance at 30 and 100 pmol/l (p less than 0.05). This potentiation was also studied in vivo. The right hind-limbs of rats weighing 150 g were infused intra-arterially by an osmotic minipump with graded doses of IGF-I (0.1 to 0.4 nmol/day) and/or PTH (0.27 nmol/day) for 7 days. The rate of trabecular bone apposition (formation) was measured by double tetracycline labelling and compared with the contralateral uninfused limb which acted as the control. Histomorphometric data revealed that neither IGF-I nor PTH alone had a significant effect on trabecular bone apposition rate compared with control limbs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of tumor necrosis factor on bone formation in vitro

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

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.     

Stimulation of bone matrix apposition in vitro by local growth factors: a comparison between insulin-like growth factor I, platelet-derived growth factor, and transforming growth factor beta

Many recent in vitro studies have shown effects of insulin-like growth factor I (IGF I), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF beta) on the proliferation and differential functions of bone-forming osteoblasts; however, the question whether these factors might ultimately lead to a net increase or decrease in bone formation has been difficult to assess. In this study, we have used an autoradiographic method based on the incorporation of [3H]proline into freshly synthesized bone matrix to determine the overall effects of these factors on bone matrix apposition in 21-day-old fetal rat calvariae. IGF I, PDGF, and TGF beta increased bone matrix apposition in a dose-dependent manner up to 2-fold within 48 h. In addition, they partially or completely reversed the inhibition of bone matrix apposition observed with PTH. Exogenously added TGF beta was significantly more potent than equimolar concentrations of PDGF or IGF I in stimulating bone formation. Matrix apposition was greatest when IGF I, PDGF, and TGF beta were added simultaneously to the culture medium, indicating that these factors can enhance each other in stimulating bone formation. In conclusion, our results provide direct evidence that IGF I, PDGF, and TGF beta are capable of stimulating bone formation in vitro.     

Repeatedly passed FRTL-5 rat thyroid cells can develop insulin and insulin-like growth factor-I-sensitive cyclooxygenase and prostaglandin E2 isomerase-like activities together with altered basal and thyrotropin-responsive thymidine incorporation into DNA

Repeatedly passed or aged rat FRTL-5 thyroid cells develop a high level of basal [3H]thymidine incorporation into DNA and a reduced response to TSH in medium containing 5% serum and insulin (5H medium). The basal [3H]thymidine incorporation into DNA of aged cells can exceed the TSH-induced increase in earlier passages of the same cell line (fresh cells) and the TSH response decreases from more than 10-fold above basal in fresh cells to less than 2-fold in aged cells. This change is not associated with a loss of the diploid karyotype, a change in basal cAMP levels, or a change in dependence on TSH for cell growth. Attenuation of the TSH response in the [3H]thymidine incorporation assay is more evident than the reduced effect of TSH on cAMP levels or iodide transport; moreover, the TSH effect on cAMP levels does not correlate with that on [3H] thymidine incorporation as a function of hormone concentration. The high basal activity in [3H]thymidine incorporation into DNA in aged cells is due to an increased responsiveness to insulin, insulin-like growth factor-I (IGF-I), or serum. Thus, removal of serum and insulin from the medium eliminates the high basal [3H]thymidine incorporation into DNA, and this activity is restored by insulin or IGF-I in a concentration-dependent manner. The increased responsiveness of aged cells to insulin or IGF-I is inhibited by indomethacin or hydrocortisone and is associated with insulin or IGF-I, but not TSH, stimulation of cyclooxygenase and prostaglandin E2 (PGE2) isomerase-like activity. Fresh cells, in contrast, require TSH plus insulin or IGF-I to increase these activities. Increased responsiveness of cyclooxygenase activity to insulin or IGF-I in aged cells reflects at least in part an increase in cyclooxygenase mRNA levels. We suggest that insulin/IGF-I stimulation of PGE2 production leads to the high basal thymidine incorporation into DNA in aged cells maintained in TSH-depleted (5H) medium; the reduced stimulation by TSH of cAMP content or iodide uptake may reflect PG inhibition (negative feedback regulation) of cAMP production.     

The interaction of heparin and basic fibroblast growth factor on collagen synthesis in 21-day fetal rat calvariae.

We examined the interactions of the glycosaminoglycan, heparin, and recombinant human basic fibroblast growth factor (bFGF) on collagen synthesis in 21-day fetal rat calvariae. In calvariae treated for 96 h, heparin (25 micrograms/ml) and bFGF (10(-9) M) inhibited collagenase-digestible protein (CDP) labeling by 52 and 60% of control, respectively, and the combination further inhibited CDP labeling. Inhibition of CDP labeling by heparin (25 micrograms/ml) or bFGF (10(-9), 10(-8) M) was similar in the presence or absence of aphidicolin (30 microM) an inhibitor of cell replication. Heparin selectively inhibited CDP labeling in the osteoblast rich central bone but bFGF alone or in combination with heparin inhibited CDP labeling both in the periosteum and central bone. Heparin and bFGF alone decreased steady state levels of alpha 1(I)procollagen messenger RNA (mRNA) at 24 h and the combination further decreased mRNA levels. A high concentration of insulin-like growth factor-1 (IGF-1, 3 x 10(-8) M) reversed the inhibitory effect of heparin on DNA synthesis and CDP labeling. In contrast, IGF-1 could not reverse the inhibitory effects of bFGF on CDP labeling but enhanced the stimulatory effects of bFGF on thymidine incorporation into DNA. We conclude that the inhibitory effects of heparin and bFGF on CDP are independent of effects on cell replication. We further conclude that both heparin and bFGF inhibit collagen synthesis at a pretranslational site since they decreased procollagen mRNA levels in osteoblasts. However, the inhibition of collagen synthesis by heparin and bFGF appears to involve divergent pathways since exogenous IGF-1 could overcome the effect of heparin but not bFGF on collagen synthesis.     

Effects of prostaglandin F2 alpha on bone formation and resorption in cultured neonatal mouse calvariae: role of prostaglandin E2 production

Although most studies show that prostaglandin E2 (PGE2) is the most potent and effective of the prostanoids in bone, recent data in cell culture suggest that PGF2 alpha may have unique effects, particularly on cell replication. The present study was undertaken to compare the effects of PGF2 alpha and PGE2 in cultured neonatal mouse parietal bones by simultaneous measurement of bone resorption as release of previously incorporated 45Ca, bone formation as incorporation of [3H]proline into collagenase-digestible (CDP) and noncollagen protein, and DNA synthesis as incorporation of [3H]thymidine. PGF2 alpha was less effective than PGE2 as a stimulator of bone resorption, and its effects were partially inhibited by indomethacin and markedly inhibited by glucocorticoids. In contrast, the resorptive response to PGE2 was unaffected by indomethacin and only partially inhibited by cortisol. PGF2 alpha had little effect on bone formation, in contrast to the biphasic effect of PGE2, which inhibited labeling of CDP in the absence of cortisol and stimulated CDP labeling in the presence of cortisol. PGF2 alpha increased thymidine incorporation into DNA, but the effect was smaller than that of PGE2 and was inhibited by indomethacin. These observations suggested that PGF2 alpha might act in part by stimulating PGE2 production. By RIA, PGE2 concentrations were increased in the medium of bones treated with PGF2 alpha, and this increase was blocked by indomethacin. By HPLC, bones prelabeled with [3H]arachidonic acid showed an increase in labeled PGE2 release, and RIA showed an increase in PGE2 after PGF2 alpha treatment. These results indicate that PGF2 alpha is a relatively weak agonist in bone compared to PGE2 and that some of the effects of PGF2 alpha on bone resorption, formation, and cell replication may be mediated by an increase in endogenous PGE2 production.